An improved fetal hemoglobin for genetic correction of sickle cell disease

ABSTRACT

Methods and compositions disclosed herein generally relates to methods of determining minimum hematopoietic stem cell (HSC) chimerism and gene dosage for correction of a hematopoietic disease; in particular, in in vivo models. The invention also relates to modified lentiviral expression vectors for increasing a viral titer and various methods for increasing such titers as well as expression vectors capable of enhancing such titers. The invention also relates to CHS4 chromatin insulator-derived functional insulator sequences. The invention also relates to methods for genetic correction of diseases or reducing symptoms thereof, such as sickle cell anemia or β-thalassemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority 35 U.S.C. §119(e)to U.S. Provisional Application No. 61/933,788, filed on Jan. 30, 2014,which is hereby incorporated by reference in its entirety

GOVERNMENT RIGHTS

This invention was made with government support under HL070595,HL70135-01, HL073104, and HL06-008 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention disclosed herein generally relates to methods ofdetermining minimum hematopoietic stem cell (HSC) chimerism and genedosage for correction of a hematopoietic disease; in particular, in anin vivo model. The invention also relates to modified SIN lentiviralexpression vectors for increasing a viral titer and various methods forincreasing such titers as well as expression vectors capable ofenhancing such titers. The invention also relates to CHS4 chromatininsulator-derived functional insulator sequences to help increase thesafety of integrating vectors and to increase expression. The inventionalso relates to methods for genetic correction of diseases or reducingsymptoms thereof, such as sickle cell anemia and β-thalassemia. Theinvention further relates to various expression vectors capable ofgenetically correcting sickle cell anemia or β-thalassemia, or reducingsymptoms thereof.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Genetic Correction and Vector Design

Successful genetic correction of diseases, mediated by hematopoieticstem cells (HSCs), depends upon stable, safe, targeted gene expressionof therapeutic quantities. Expression vectors are central to the processof genetic correction and consequently the subject of considerableresearch. Although significant advances in vector design have improvedthe efficacy of gene therapy, certain key obstacles have emerged asbarriers to successful clinical application. Among those obstacles,vector genotoxicity is among the most formidable, as evidenced by theoccurrence of gene therapy related leukemia in patients in X-SCIDtrials, as disclosed herein. As a result, gamma-retroviral vectors andlentiviral vectors have been modified to a self-inactivating (SIN)design to delete ubiquitously active enhancers in the U3 region of thelong terminal repeats (LTR) (as disclosed herein). SIN design has beenimproved upon to increase vector titers. Several methods of improvingtransgene expression have been subsequently employed.

As an added measure of stabilizing expression, many vectors are nowdesigned with chromatin insulating elements that reduce chromatinposition effects. While these insulators can improve the safety andexpression profiles of certain vectors, in some cases an undesirableside effect is decreased titers compared to non-insulated versions.Custom insulators have been designed that provide optimal insulationwithout lowering titers.

Thus, there is a need in the art for improved expression vector design,aimed at safely stabilizing the expression of transgenes, whilemaintaining clinically relevant viral titers.

Determining Critical Parameters of Correction in Sickle Cell Anemia

Expressing a tremendous amount of fetal/antisickling hemoglobin willundoubtedly correct disease, as has been demonstrated, but is notpractically possible in a clinical setting. As an example, an initialgene therapy for adenosine deaminase (ADA) deficiency was performedusing no conditioning, and was not therapeutic, even though fewgene-marked stem cells engrafted, and a selective advantage togene-corrected lymphocytes was evident upon withdrawal of ADA (asdisclosed herein). In a subsequent trial, 4 mg/kg busulfan was usedbefore transplantation, as conditioning, resulting in adequategene-corrected stem cell dose and gene-modified T cells (as disclosedherein). Thus, there is a need in the art to establish methods ofdetermining thresholds for genetic correction before embarking onclinical studies.

SUMMARY OF THE INVENTION

Methods and composition described herein are provided by way of exampleand should not in any way limit the scope of the invention.

Embodiments of the invention encompass mutated human gamma-globin genes.In some embodiments, the mutated human gamma-globin gene can encode aprotein including SEQ ID NO:1. In some embodiments, the mutated humangamma-globin gene can have a sequence identity of 70% or greater to SEQID NO: 2.

Embodiments of the invention also encompass methods of using a mutatedhuman gamma-globin gene encoding a protein including SEQ ID NO:1 togenetically correct sickle cell anemia or β-thalassemia or reducesymptoms thereof, the method including identifying a subject in need oftreatment for sickle cell anemia or β-thalassemia; transfectingautologous hematopoietic stem cells (HSCs) with a modified lentivirusincluding the mutated human gamma-globin gene encoding a proteinincluding SEQ ID NO:1; and transplanting the transfected HSCs into thesubject.

In some embodiments, the subject is a human subject. In someembodiments, the subject is treated with reduced intensity conditioningprior to transplantation.

In some embodiments, the modified lentivirus further includes aheterologous polyA signal sequence downstream from a viral 3′ LTRsequence in a standard SIN lentiviral vector backbone; and one or moreUSE sequences derived from an SV40 late polyA signal in a U3 deletionregion of a standard SIN lentiviral vector backbone. In someembodiments, the modified lentivirus further includes one or moreflanking CHS4-derived reduced-length functional insulator sequences. Insome embodiments, the modified lentivirus further includes a beta-globinlocus control region. In some embodiments, the modified lentivirusfurther includes an erythroid lineage specific enhancer element.

In some embodiments, post-transplantation fetal hemoglobin exceeds atleast 20%; F cells can be at least ⅔ of the circulating red blood cells;fetal hemoglobin per F cells can be for at least ⅓ of total hemoglobinin sickle red blood cells; and at least 20% gene-modified HSCs canre-populate bone marrow of the subject.

Embodiments of the invention also encompass lentiviral expressionvectors capable of genetically correcting sickle cell anemia orβ-thalassemia or reducing symptoms thereof, includes a mutated humangamma-globin gene encoding a protein in SEQ ID NO:1. In someembodiments, the lentiviral expression vector, further includes aheterologous polyA signal sequence downstream from a viral 3′ LTRsequence in a standard SIN lentiviral vector backbone; and one or moreUSE sequences derived from an SV40 late polyA signal in a U3 deletionregion of a standard SIN lentiviral vector backbone. In someembodiments, the lentiviral expression vector further includes one ormore flanking CHS4-derived reduced-length functional insulatorsequences. In some embodiments, the lentiviral expression vector furtherincludes one or more elements of a beta-globin locus control regioncloned in reverse orientation to a viral transcriptional unit. In someembodiments, the lentiviral expression vector further includes anerythroid lineage specific enhancer element.

BRIEF DESCRIPTION OF THE FIGURES

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 depicts titers from the standard and gutted SIN-LV (a) Aschematic representation of SIN lenti-proviruses. sSIN-GFP, sBG-6 andsFIG are SIN-LV carrying GFP, the β-globin gene (BG) or the FanconiAnemia A cDNA,-IRES-GFP respectively. dsSIN-GFP, sBG-1 and ds-FIG aretheir gutted counterparts. SD=splice donor. SA=splice acceptor. ψpackaging sequence. cPPT: central poly purine tract. The gag (360 bp)and the env fragment containing the RRE (˜850 bp) are indicated. (b) Theviral obtained after infection of MEL cells and analysis for GFP andhβ-globin expressing cells with different iterations of the “SIN”design. Titers are expressed as IU/mL of concentrated supernatant (n=3).

FIG. 2 depicts BG SIN-LV constructs. A schematic representation of 10SIN-lentiviral proviral forms (sBG-1 to sBG-10). All the vectors containBG (HS2, 3, and 4 elements of the LCR, the (β-promoter and gene) andcPPT. Gag (630 bp or 360 bp), RRE, env fragments are shown. * indicatesa point mutation that disrupts the SA.

FIG. 3 depicts viral titers of BG SIN-LV (a) Viral supernatants of sBG-1to sBG-10 SIN lentiviral vectors were concentrated 1400-fold and titeredon MEL cells by monitoring for β-globin positive cells by flow cytometry(n=4). (b) Fold increase in titers with inclusion of cis-elements. Thetiters were normalized to that of the completely gutted vector (sBG-1),which was considered 1. The sBG-6 design showed a marked increase intiters.

FIG. 4 depicts effect of LV cis-elements on the provirus stability andexpression. (a) Proviral integrity: Southern blot analysis of MEL cellstransduced with sBG-127 to sBG-10, restricted with AflII that cuts inthe viral LTRs, and probed with a hβ-globin fragment. All SIN vectorsare transmitted stably. (b) Expression of hβ-globin in MEL cells: dotplot analysis of sBG-1 to sBG-10 transduced MEL cells from onerepresentative experiment; MFI are indicated in the upper right cornerof the dot-plot.

FIG. 5 depicts vRNA transcripts in packaging cells. Northern blotanalysis of (A) total RNA from 293T packaging cells transfected with SINLV plasmids and probed with a ³²P labeled hβ-globin fragment. Lowerpanel shows the same blot hybridized with an 18S probe as loadingcontrol. A full length band of the expected size is visible for all thevectors. * indicate vectors in which SA is present and both full lengthand spliced bands are visible. A small schematic of the vectorcis-sequences are shown above the vector lanes to depict the Ψ packagingsequence; R: RRE; SA: Splice Acceptor in the env fragment; SG: short gagfragment (360 bp); LG: long gag fragment (630 bp) in vectors. (B)Cytoplasmic RNA for vectors with and without RRE from the sameexperiment shown in panel A, showing the efficiency of vRNA export intothe cytoplasm. The phosphoimager quantified ratios cytoplasmic/total areshown in FIG. 6E.

FIG. 6 depicts packaging of vRNA into virons (a) A representative dotblot analysis on vRNA extracted from sBG series of virus supernatantsshowing that the amount of vRNA is proportional to infectious titers.Virus was made from all ten vectors and concentrated identically asdescribed and the dot-blot was probed with a β-globin fragment.NC=negative control. Four different dilutions for each vector wereloaded in duplicate in the representative experiment shown. A total ofthree experiments were performed (B) Phosphoimager counts obtained onthe 28 dot blot shown in panel (A). (C) Relative quantification of vRNAfrom all three experiments. (D) p24 activity in concentrated virus fromall vectors (n=2). (E) Ratio of Cytoplasmic/Total RNA from 2 NorthernBlot Analysis (NB) in Packaging Cells (The ratio cytoplasmic/total RNAwas normalized to the value for the completely gutted vector lacking theRRE (SBG-1) and to 18S RNA (for loading) in two independent experiments.Analogous vectors with and without RRE are marked as I, II and III toallow ready comparisons).

FIG. 7 depicts vector constructs and experimental design. A.Self-inactivating (SIN) lentiviral vector carrying the hβ-globin geneand the HS2, HS3 and HS4 of the locus control region is shown as sBG.Using this backbone, a series of vectors were generated to incorporateeither the cHS4 59 250 bp core, 2 tandem repeats of the core, 5′ 400 bpor 59 800 bp of cHS4, and the full-length 1.2 Kb cHS4 insulator. VectorssBG400Sand sBG800S carry in addition to the core inert DNA spacers from1 bacteriophage. B. Schema of In vitro and in vivo analyses: MEL cellswere transduced with various vectors to derive single copy MEL clonesand hβ-globin expression and ChIP analysis was performed indifferentiated clones. In vivo analysis was done using vector transducedHbb^(th3/+) donor LSK cells transplanted into lethally irradiatedHbb^(th3/+) recipients and analyzed at 6 months post-transplant.Secondary transplants were performed for CFU-S analysis. C.Representative FACS plot showing hβ-globin-expressing cells (% hβ+) foruninsulated (sBG, green) and insulated (sBG-I, Pink) single copy MELclone with coefficient of variation (CV) of expression shown by arrows.

FIG. 8 depicts human β-globin expressing cells in MEL clones. A.Proportion of hβ-globin-expressing cells (% hβ+) in MEL clones. Eachcircle represents an individual single copy MEL clone. B. CV values ofhβ-globin expression of each clone. The means are represented with ahorizontal line and the mean 6 SEM of % hβ+ MEL cells and CV ofhβ-globin expression for each vector are indicated in the box above.Filled circles represent representative clones picked for ChIPanalysis. * P<0.05 by ANOVA, as compared to sBG.

FIG. 9 depicts human β-globin expression in RBCs and single copysecondary CFU-S. A. Representative FACS histograms showing (% hβ+ RBCare indicated within the histogram). B. Cumulative data on thepercentage of hβ+ RBCs normalized to vector copy. C. The coefficient ofvariation (CV) of hβ expression in RBCs. D. Cumulative data on % hβ+cells/CFU-S. Each circle represents an individual single integrantCFU-S. E. The CV of hβ expression in the individual CFU-S. Numbers abovebar diagrams represent mean 6 SEM and values significantly differentfrom controls by ANOVA are marked by an asterisk. * P<0.05; ** P<0.01.

FIG. 10 depicts chIP analysis showing the active and repressive histonemarks on the 5′ 250 bp cHS4 core and the hβ promoter in MEL cell clones.A. Map of the proviral form of the vector. Arrows show the position ofthe primer pairs used for PCR and qPCR; and the lines representinsulator fragments. B-C. ChIP with antibodies against control IgG,acH3, acH4, H3K4-me2, H3K9-me3 and H3K27-me3 and semiquantitative PCRprimers to the β-globin promoter region D-F ChIP with antibodies to AcH3and AcH4 (D), H3K4-me2 (E); H3K9-me3 and H3K27-me3 (F) followed by qPCRusing primers amplifying cHS4 core (left panels) and hβ-globin promoter(right panel) on pooled clones (shown in FIG. 2A). *P<0.05; **P<0.01.

FIG. 11 depicts human β-globin expression in mice. A. RBC parameters,reticulocytes and vector copies. Values represent means±SEM.Hb=hemoglobin, MCV=mean corpuscular volume, MCHC=mean corpuscularhemoglobin concentration, vector copy=vector copies in leukocytes byqPCR. B. HPLC analysis of human β-globin protein in blood lysates as apercentage of total hemoglobin [hβ-mα/(hβ-mα+mβ-mα)]. Data is normalizedto vector copy/cell in leukocytes. *P<0.05; **P<0.01.

FIG. 12 depicts effect of 3′400 bp region of the cHS4 insulator. A.Vector design of sBG^(3′400) vector. The full length cHS4 is shown forcomparison. B-C. Proportion of hβ+ cells (B) and the coefficient ofvariation of hβ expression of sBG^(3′400) (C) in MEL clones. Each circlerepresents a single integrant MEL clone. The means are represented witha horizontal line and the mean±SEM are represented in the figure. D-E.The percentage of hβ-globin+ RBC (D), and the CV of hβ expression (E) inmice. F-G. hβ-globin-expressing cells (F) and the CV of hb expression(G) in single copy CFU-S following secondary transplant. Each circlerepresents individual CFU-S. Mean±SEM and P-values are shown. * P<0.05;**P<0.01; *** P<0.001.

FIG. 13 depicts effect of the combination of the 5′ core with the 3′ 400bp regions of the cHS4 insulator. A. Vector design of sBG⁶⁵⁰. The fulllength cHS4 is shown for comparison. B. Proportion of hβ+ cells and C.CV of hb-globin expression in sBG⁶⁵⁰ MEL clones. Each circle representsa single copy MEL clone. The means are represented with a horizontalline and the mean±SEM is indicated above each group. D. Percentage ofhbglobin expressing RBC in transplanted mice. E. Percentage hβ-globinexpressing cells in single copy CFU-S from secondary mice. F-G. ChIPactive and repressive chromatin followed by semiquantitative PCR (F) orqPCR (G) of the cHS4 core region or the hβ-globin promoter region.

FIG. 14 depicts chromatin patterns over the 3′400 bp and its interactionwith the 5′ core region. A. A map of 3′LTR showing location of fulllength 1.2 kb insulator and the position of primers used in ChIPanalysis. Vectors tested with the indicated regions of the cores aredepicted beneath map B ChIP with antibodies to AcH3 and AcH4, H3K4-me2and H3K9-me3 and H3K27-me3 followed by a semiquantitative PCR of the3′400 region in sBG^(3′400), sBG⁶⁵⁰, sBG-I provirus. C-D ChIP withantibodies to USF-1 and CTCF followed by semi-quantitative PCR (C) orqPCR (D) for the core region. E-F ChIP with antibodies to USF-1 and CTCFfollowed by semi-quantitative PCR (C) or qPCR (D) for the 3/400 bpregion of the sBGC, sBG^(3′400), sBG⁶⁵⁰ and sBG-I provirus in pools ofthree single copy MEL clones. (G) Figure S1 Representative histograms(FACS) showing hb expressing cells in mock, sBG, sBGC, sBG2C, sBG400 andsBGI sBGI single copy CFU-S. The % of hβ+ cells are indicated within thehistogram. (H) Human β-globin messenger RNA (mRNA) expression in singlecopy secondary CFU-S of sBG, sBGC, sBG2C,sBG400 and sBG-I by qPCR.Murine a-globin expression served as the internal control against whichhβ-globin expression was normalized. P values are shown in the figure.** indicates P<0.01. (I) The primers and probes used in chromatinimmunoprecipitation (ChIP) is shown. ‘F’ represents forward primer and‘R’ represents reverse primer. (J) Insertional site analysis on singlecopy MEL clones from uninsulated sBG and insulated sBG-I vector withgene hits according to http://genome.ucsc.edu.

FIG. 15 depicts viral titers of lentiviral vectors with inserts into the3′LTR were inversely proportional to the length of the LTR insert. (A)Schematic representation of the lentiviral vectors. All vectors werebased on sBG, a SIN lentiviral vector carrying the β-globin gene,β-globin promoter and the locus control region elements H52, HS3 andHS4. Different fragments of the cHS4 site were inserted in the U3 regionof the sBG 3′LTR (shown above the sBG vector). Similar sized insertswere made by replacing the region downstream of cHS4 core with inert DNAspacers from the lambda phage DNA (shown below the sBG vector). (B)Viral titers of insulated vectors decreased as the length of theinsulator insert increased. Titers reflect concentrated virus madeconcurrently for all vectors in each experiment (n=4). All titers weresignificantly lower than the titers of the control vector sBG (p<0.01;1-way ANOVA). (C) Titers fell with insertion of increasing length of aninert DNA spacer downstream of the core. Titers of insulated lentivirusvectors (hatched bars) are similar to those containing inert DNA spacersin the LTR (open bar) in four independent experiments. The titers of sBGwith a 400 bp spacer were slightly higher (* p<0.05). (D) The sBG^(2C)vector, carrying tandem repeats of the cHS4 core recombined with highfrequency. A schematic representation of the vectors sBG-I and sBG^(2C)proviruses, when intact, or when the core elements recombine with lossof one or two cores with the region probed and restriction site of theenzyme used (AflII) is shown. The size of the expected band is shownadjacent to each vector cartoon. The right panel is the Southern blotanalysis showing a single 8 Kb expected band for sBG-I transduced MELcell population, and two bands in the sBG^(2C) transduced MEL cellpopulation, representing sBG^(2C) with either loss of one or both cores.

FIG. 16 depicts similar amounts of viral RNA were produced from theinsulated and uninsulated vectors in packaging cells. Northern blotanalysis on the 293T packaging cells after transfection with sBG andsBG-I vectors showed the expected length viral RNA. The membrane washybridized with a ³²P labeled p-globin probe (top panel) and 18S (bottompanel) as a loading control. An expected 7.3 Kb and 8.5 Kb bandcorresponds to sBG and sBG-I viral RNA were detected. The 18S and 28SrRNA was non-specifically probed with this probe. No extraneousrecombined bands were detected with either vector. The phosphoimagerquantified ratios of viral RNA and 18S rRNA of both vectors are listedbelow the lanes and show no difference in the amount of v-RNA betweenthe two vectors.

FIG. 17 depicts virus production was not impaired by insertion of cHS4in the 3′LTR (A) Reverse transcriptase activity in sBG and sBG-I viralsupernatants is similar (23±5 vs. 27±3; n=3, p>0.5). (B) p24 levelsdetected in the concentrated viral preparation is the same with sBG andsBG-I. (2.9±0.5×10⁵ versus 1.7±0.5×10⁵; n=3, p>0.1) (C) Dot-Blotanalysis of viral RNA extracted from sBG and sBG-I viral supernatantshows similar amounts of viral RNA packaged into virions in bothvectors. Note that 4 different dilutions of viral RNA were loaded induplicate for the two vectors. The membrane was hybridized with a ³²Plabeled p-globin probe. Only one of two representative experiments isshown. (D) Phosphoimager quantification of two independent experimentswas plotted and showed similar amounts of viral RNA in sBG and sBG-Ivirions (1.9±0.7×10⁶ vs. 1.9±0.6×10⁶n=2, p>0.5).

FIG. 18 depicts kinetic of reverse transcription and nucleartranslocation in lentivirus vector carrying insulator element in theLTR. In panel (A) a schema of the lentivirus reverse transcription andnuclear translocation process is illustrated. On the right a summary ofq-PCR assays performed to analyze several steps of the process. Thinline: RNA; thick line: DNA. Open boxes: polypurine tract (PPT). Opencircle: priming binding site (PBS). The 3′ LTR DNA insert is illustratedin the first strand transfer diagram. The positions of the q-PCR assaysare shown. DNA from MEL cells after infection with sBG and sBG-I viruswas collected at different time points after infection and analyzed byqPCR. Solid line: sBG. Dashed line: sBGI. (B) Kinetic of reversetranscription before the first strand transfer (R/U5) shows nodifference between the two viruses. (C-D) After the first strandtransfer (U3/R and Psi) there is a decrease in reverse transcriptionefficiency in presence of the insulator. (n=3).

FIG. 19 depicts insertion of cHS4 in the LTR affected viral integration.Linear viral cDNA circularizes and is the form that integrates; 1-LTRand 2-LTR circles represent abortive integration products fromhomologous recombination and non-homologous end joining, respectively.The 1- and 2-LTR circles are therefore used as markers of nucleartranslocation. (A) There are reduced 2-LTR circles, analyzed by qPCR onDNA extracted from MEL cells infected at different times after infectionwith virus suggesting reduced nuclear translocation or non-homologousend-joining. (B) Southern blot analysis of MEL cells 72 h afterinfection with same amount of sBG and sBG-I virus. StuI digestion ofgenomic DNA allows identification of 1-LTR circles, 2-LTR circles,linear DNA and integrated DNA (a smear) for sBG and sBG-I. Expected bandsizes are shown for both vectors. While linear, 1- and 2-LTR circles areseen in the sBG lane, no linear DNA or 2-LTR circles are detected in thesBG-I lane. However, 1-LTR circles are almost as prominent as in the sBGlane. The relative ratios of linear, 1- and 2-LTR circles suggestincreased recombined abortive integration products with the sBG-Ivector, and hence result in inefficient integration. (C) sBG and sBGItransduced MEL cells show intact proviral integrants (7.5 Kb and 8.0 Kbrespectively). There was an 8-fold difference in phosphoimager counts ofthe two bands. Vector copy number per cell was also quantified by qPCRand is depicted below the lanes.

FIG. 20 depicts hypothesis of mechanism by which insulator sequencedecrease viral titer. In wild type HIV, linear cDNA moleculestranslocate to the nucleus where a small percentage undergoesrecombination and end joining ligation to form 1- and 2-LTR circles,respectively. Only the linear form is the immediate precursor to theintegrated provirus. In the case of insulated LV vectors, it is shown anincrease in 1LTR circle formation, due to the presence of a larger U3sequence that could facilitate an increase in homologous recombination.This process depletes the amount of viral DNA available for integrationas well as the amount of 2-LTR circle formation, as shown herein. Thedecreased amount of DNA available for integration could explain the lossin titers for lentivirus vector carrying large inserts in the LTR. (B) Afurther addition of a 1.2 Kb PGK-MGMT internal cassette to the BG-Ivector, termed BGM-I, did not reduce the titers any further (C) Anoptimized vector design results in reasonable virus titers without lossof insulator activity. A 650 bp sequence of cHS4, optimized forinsulator activity through a structure function analysis. A vectorcontaining this 650 bp fragment (sBG650), was found to have ˜2-foldlower titers than the uninsulated vector sBG (n=3). (D) PCR for Presenceof 3′LTR Inserts in Proviruses Derived from Single Copy MEL Clones ShowsStable Transmission of all Inserts Except those Present as TandemRepeats MEL cells were cloned from pools with ≦5% gene transfer. Singlecopy clones were detected using β-globin primers and confirmed by a qPCRusing primers spanning the ψ region. PCR with primers spanning the 250bp core was performed in the single copy clones, as these core sequenceswere common to all vectors. The 1.2 Kb cHS4 insert in the sBG-I vectorwas further confirmed by PCR primers spanning the 5′ core and the 3′ endof cHS4. (E) PCR primers.

FIG. 21 depicts sG^(b)G mice that underwent transplantation aftermyeloablative conditioning have high HbF production that is stable andsustained in primary and secondary mice. sG^(b)G mice that were fullychimeric for donor RBCs were analyzed at different time points. Theproportion of HbF (A) and F cells (B) in blood of individual mice, asdetermined by ion-exchange HPLC and FACS analysis, respectively, isshown at different time points after primary and secondarytransplantations. (C) The amount of HbF in blood directly correlatedwith the proportion of F cells. (D) The amount of HbF produced wasdirectly in proportion to the vector copy number in bone marrow. Eachsymbol represents one mouse (and consistently depicts the sameparticular mouse in all the panels). (E) Hematologic parameters ofsG^(b)G mice that underwent transplantation after myeloablativeconditioning. Hb indicates hemoglobin; MCV, mean corpuscular volume;MCH, mean corpuscular hemoglobin; RDW, red cell distribution width; Pit,platelets; pri, primary mice; and sec, secondary mice. *P valuesrepresent comparison of primary mock mice with the sG^(b)G group.Statistical comparisons of secondary mice were not made as only onesecondary mock mouse was alive at the time of analysis.

FIG. 22 depicts sG^(b)G mice that underwent transplantation aftermyeloablative conditioning, which resulted in correction of hematologicparameters that correlated with the HbF expression. There was sustainedreduction in reticulocytes (A), and increase in hematocrit (B) and RBCnumbers (C) over time, (D) Leukocytosis decreased with normalization ofWBC counts. Data shown represent mean (±SEM) values of sG^(b)G mice=5;•) and mice that underwent mock transplantation (n=10; O). A starrepresents mean values in BERK mice that were HSC donors for the sG^(h)Gand mock transplantations. (E-G) Decrease in reticulocytes, andincreased hematocrit and RBC numbers correlated with the proportion of Fcells in individual mice. (H) WBC counts decreased but normalized whenthe F cells exceeded 60%. WBC counts, counted on an automated analyzer,were representative of circulating leukocytes, since only occasionalnucleated RBCs were seen in peripheral smears. Each data point/symbol inpanels E-H represents one sG^(b)G mouse and symbols for individual micehave been kept consistent, to trace individual mice. A star representsmean values in BERK mice that were HSC donors for the sG^(b)G and mocktransplantations.

FIG. 23 depicts sG^(b)G mice that underwent transplantation aftermyeloablative conditioning, which resulted in correction of functionalRBC parameters in primary and secondary mice. (A) Peripheral bloodsmears showing numerous irreversibly sickled cells (ISCs) in a mousethat underwent mock transplantation and a paucity of ISCs in a sG^(b)Gmouse. (B) Quantification of ISCs in peripheral blood smears of BERKmice that did not undergo transplantation (n=5), mock mice (n=3), andsG^(b)G mice (n=5). (*P<0.05; **P<0.01). (C) Deoxygenation of bloodinduces sick-ling of RBCs in a mock mouse; sickling is largely absent ina sG^(b)G mouse. (D) Quantification of sickle RBCs upon graded hypoxia(by tonometry) in the sG^(b)G mice (•), compared with mock mice (O). (E)RBC deformability by LORCA analysis in sG^(b)G, mock, and normal mice(C57, circle with x in center) analyzed at 18 weeks in primarytransplant recipients. Similar data were seen in secondary recipients.Flow at low (3 Pa) and high (28 Pa) shear stress is represented byshaded areas. (F) RBC half-life (determined by in vivo biotin labeling)in the sG^(b)G mice, mock/BERK mice, and normal mice after primarytransplantations. Similar results were seen in secondary recipients. (G)Correction of organ pathology in sG^(b)G mice that underwenttransplantation with myeloablative conditioning. 2+ liver infarctionindicates 2 to 3 infarctions/section; 3+ liver infarction, more than 3infarctions/section; and E-M, extramedullary. Mild congestion of thespleen vessels with sickle RBCs is seen when splenic architecture isrestored. This is not noted when the splenic architecture is effaced byextramedullary erythropoiesis. Splenic erythroid hyperplasia: severe iscomplete obliteration of splenic follicles; moderate, more than 1follicle present/section; and mild, preservation of follicles withevidence of erythroid islands. Bone marrow: normal erythropoiesisindicates M/E=5:2; mild erythroid hyperplasia, M/E=2:1; moderateerythroid hyperplasia, M/E=1:1; and severe erythroid hyperplasia,M/E=1:3. Bone marrow erythropoiesis expressed as myeloid-erythroid ratio(M/E). Numbers in parentheses indicate the histologic feature seen inthe number of mice/total number of mice analyzed in that group.

FIG. 24 depicts HbF expression and functional correction in sG^(b)G micethat underwent transplantation after reduced-intensity conditioning,separated into 2 groups: mice with HbF of 10% or more (sG^(b)G>10) andmice with HbF of less than 10% (sG^(b)G<10). (A) HbF in individual BERKmice 18 weeks after transplantation of sG^(b)G-transduced BERK HSCs,after reduced-intensity conditioning. (B-C) Stable and high HbFexpression and F-cell repopulation in long-term survivors analyzed at 11months. (D) Box and whisker plot showing vector copy numbers insG^(b)G<10 and sG^(b)G>10 mice, with mean vector copy number denoted bythe line. Symbols in panels A through C represent mouse groups: O=mock(HbF 0%), ▾=sG^(b)G<10 (HbF<10%), and •=sG^(b)G>10 (HbF>10%). (E) Theproportion of ISCs was reduced (P<0.04) in sG^(b)G<10 mice, but wasmarkedly reduced in sG^(b)G>10 mice (P<0.001), compared with mock mice.(F) Graded deoxygenation via tonometry demonstrates significantreduction in sickling at physiologically relevant partial oxygenpressures (PO2) in sG^(b)G>10 mice, whereas sG^(b)G<10 mice RBC sickledsimilar to controls. (G-H) RBC deformability showed highly variableimprovement in deformability in sG^(b)G<10 mice. In contrast, RBCdeformability in sG^(b)G>10 mice was highly significantly improved atlow and high shear stress (P<0.001). Symbols represent mouse groups: 0,mock; ▾, sG^(b)G<10; •, sG^(b)G>10; and (circle with x in center),wild-type mice (C57BL/6). Gray shaded rectangles are representative oflow and high shear stress through microvessels and large vessels,respectively. Error bars indicate SEM.

FIG. 25 depicts correction of organ pathology in sG^(b)Ĝ10 mice thatunderwent transplantation after reduced-intensity conditioning andimproved overall survival. (A) Representative hematoxylin-eosin-stainedsections of a kidney, liver, and spleen of sG^(b)G>10 and sG^(b)G<10mice 48 to 50 weeks after reduced-intensity conditioning transplantationand a 3-month-old BERK control. Image acquisition information isavailable in supplemental data. (B) Kaplan-Meier survival curve showedsignificantly improved survival of the sG^(b)G>10 mice compared withmock/sG^(b)G<10 mice at 50 weeks. Survival at 24 weeks is denoted by adashed vertical line to compare with survival of the sG^(b)G mice in themyeloablative transplantation model, (C) Hematologic parameters ofsG^(b)G mice that underwent transplantation following reduced-intensityconditioning. Hematologic parameters and abbreviations as stated in thefigure. P values represent comparisons of mock mice with sG^(b)G≧10 at12(*), 18 (†), and 24(‡). (D) Organ pathology in sG^(b)G mice thatunderwent transplantation after reduced-intensity transplantation. E-Mindicates extramedullary; and 1+ liver infarction. 1 infarction/section.*Congestion of vessels and presence of sickle RBC in vessels. Notably,congested vessels were visible in spleens only when erythroidhyperplasia effecting splenic architecture was reduced. The terminologyused to quantify organ pathology is the same as documented in thefigure.

FIG. 26 depicts effect of HbF, F cells, and percentage HbF/F cellrequired for functional improvement in RBC survival and deformability.(A) RBC half fife. Left panel shows a representative sG^(b)G mouseinjected with biotin with biotin-labeled F cells (upper right quadrants)and non-F cells (lower right quadrants) determined by FACS. Right panelshows survival of F cells (hollow square with solid circle in center),compared with the non-F cells (Hollow circle with solid circle incenter) in sG^(b)G mice=4); wild-type mice (A); and Berkeley mice (O).(B) A cohort of sG^(b)G mice analyzed for RBC survival in vivo, basedupon the percentage of HbF/F cell. Each symbol represents a mouse groupwith HbF percentage and number of mice listed in the adjacent tablelegend. (C) All sG^(b)G and mock mice (n=34) that were analyzed for RBCdeformability were divided into groups based on proportion of F cells0%, 1% to 33%, 33% to 66%, and more than 66%, and deformability of totalRBC in these mice was plotted at low (3 Pa, Δ) and high (28 Pa, upsidedown hollow triangle) shear stress. Significantly improved deformabilityover mock controls is denoted by *(P<0.05) and **(P<0.01). Error barsindicate SEM

FIG. 27 depicts proportion of transduced HSCs in sG^(b)G mice.Proportions in the myeloablative (A) and reduced-intensity (B)transplantation models are shown. The proportion of sG^(b)G-transducedHSCs was determined by spleen colonies (30-36 colonies/mouse) byintracellular staining with HbF and HbS. Each bar represents anindividual mouse. (A) In the myeloablative transplantation model,symbols beneath each bar (representing one mouse) are consistent withthe symbols in mice labeled, (B) In the reduced-intensity group, bonemarrow was successfully aspirated from 8 mice at 24 weeks and mice werefollowed for an additional 24 weeks. The HbF expression in peripheralblood by HPLC and bone marrow copy number of the respective mice at 24weeks are labeled under each bar.

FIG. 28 shows the improvement in survival of mice following successfulgene therapy.

FIG. 29 depicts correction of thalassemia in Hbb^(th3/+) mice with thesGbG vector. Hemoglobin (FIG. 29A) and hematocrit (FIG. 29B) werecorrected to normal levels with approximately 20% HbF expression (FIG.29C). Reticulocyte counts (FIG. 29D) were also significantly lowered,showing reduced erythroid cell turnover.

FIG. 30 depicts the annotated vector map for the sG^(b)G^(M) vector.

FIG. 31 depicts the sG^(b)G^(M) vector sequence, along with the variousregions of the sequence.

FIG. 32 depicts superior HbF expression from the sG^(b)G^(M) vector. HbFexpression from the sG^(b)G^(M) vector in Berkeley sickle mice showssuperior HbF expression per vector copy, when compared to the sG^(b)Gvector (FIG. 32A). Sickle mice transplanted with sG^(b)G^(M) transducedhematopoietic stem cells show a reduction in reticulocyte count that isproportional to HbF production (FIG. 32B). Mice with 30% or more HbFproduced from the sG^(b)G^(M) vector have nearly normal reticulocytecounts. Normal reticulocyte values are depicted via the shadedrectangle.

FIG. 33 depicts HbF expression from the sG^(b)G^(M) vector as comparedto the sG^(b)G vector in sickle mice. HbF expression from thesG^(b)G^(M) vector in Berkeley sickle mice (FIG. 33A) and knock-in UABsickle mice (FIG. 33B) shows superior HbF expression per vector copy, ascompared to the sG^(b)G vector. Sickle mice transplanted withsG^(b)G^(M) transduced hematopoietic stem cells show superior correctionof anemia (FIGS. 33C-D) and reduction in reticulocytosis (FIGS. 33E-F)that is proportional to HbF production. Mice with 30% or more HbFproduced from the sG^(b)G^(M) vector have nearly normal reticulocytecounts and correction of the sickle phenotype.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Also incorporated herein byreference in their entirety include: U.S. Non-Provisional applicationSer. No. 12/928,302, filed on Dec. 6, 2010, and U.S. ProvisionalApplication No. 61/267,008, filed on Dec. 4, 2009. Also incorporatedherein by reference in their entirety is a novel human gamma-globin genevector for genetic correction of sickle cell anemia in a humanized mousemodel and critical determinants for successful correction thereof asdescribed in, “A novel human gamma-globin gene vector for geneticcorrection of sickle cell anemia in a humanized mouse model: criticaldeterminants for successful correction”. Blood (2009) 114: 1174-1185Perumbeti A, Higashimoto T, Urbinati F, Franco R, Meiselman H et al.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 3^(rd) ed., J. Wiley & Sons (NewYork, N.Y. 2001); March, Advanced Organic Chemistry Reactions,Mechanisms and Structure 5^(th) ed., J. Wiley & Sons (New York, N.Y.2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual3^(rd) ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y. 2001), provide one skilled in the art with a general guide to manyof the terms used in the present application. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. Indeed, the present invention is in no way limited to themethods and materials described.

As used herein, the term “SIN” is an abbreviation of self-inactivating.

As used herein, the term “HIV” is an abbreviation of humanimmunodeficiency virus.

As used herein, the term “GFP” is an abbreviation of green fluorescentprotein.

As used herein, the term “cDNA” is an abbreviation of complimentary DNA.

As used herein, the term “LTR” is an abbreviation of long terminalrepeat.

As used herein, the term “USE sequence” refers to an upstream sequenceelement.

As used herein, the term “polyA” is an abbreviation of polyadenylation.

As used herein, the term “cHS4” is an abbreviation of chickenhypersensitive site-4 element.

As used herein, the term “HSC” is an abbreviation of hematopoietic stemcells.

As used herein, the term “GOI” is an abbreviation of gene of interest.

As used herein, the term “HbF” is an abbreviation of fetal hemoglobin.

As used herein, the term “RBC” is an abbreviation of red blood cell. Asused herein, the term “IDUA” is an abbreviation of alpha-L-iduronidase.

As used herein, the term “LCR” is an abbreviation of locus controlregion.

As used herein, the term “subject” refers to any member of the animalkingdom. In some embodiments, a subject is a human patient.

As used herein, the terms “treatment,” “treating,” “treat,” “correct,”and the like, with respect to a specific condition, refer to obtaining adesired pharmacologic and/or physiologic effect. The effect can beprophylactic in terms of completely or partially preventing a disease orsymptom thereof and/or can be therapeutic in terms of a partial orcomplete cure for a disease and/or adverse effect attributable to thedisease. “Treatment,” as used herein, covers any treatment of a diseasein a subject, particularly in a human, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease and/or relieving one or moredisease symptoms. “Treatment” can also encompass delivery of an agent oradministration of a therapy in order to provide for a pharmacologiceffect, even in the absence of a disease or condition. The term“treatment” is used in some embodiments to refer to administration of acompound of the present invention to mitigate a disease or a disorder ina host, preferably in a mammalian subject, more preferably in humans.Thus, the term “treatment” can include includes: preventing a disorderfrom occurring in a host, particularly when the host is predisposed toacquiring the disease, but has not yet been diagnosed with the disease;inhibiting the disorder; and/or alleviating or reversing the disorder.Insofar as the methods of the present invention are directed topreventing disorders, it is understood that the term “prevent” does notrequire that the disease state be completely thwarted (see Webster'sNinth Collegiate Dictionary). Rather, as used herein, the termpreventing refers to the ability of the skilled artisan to identify apopulation that is susceptible to disorders, such that administration ofthe compounds of the present invention can occur prior to onset of adisease. The term does not mean that the disease state must becompletely avoided.

As used herein, the terms “mutated,” “mutation,” “mutant,” and the like,refer to a change in a sequence, such as a nucleotide or amino acidsequence, from a native, wild-type, standard, or reference version ofthe respective sequence, i.e. the non-mutated sequence. These terms canrefer to one or more mutated genes, such as deoxyribonucleic acids,ribonucleic acids, and the like, or one or more mutated gene products,such as proteins. A mutated gene can result in a mutated gene product. Amutated gene product will differ from the non-mutated gene product byone or more amino acid residues.

In some embodiments, a mutated gene which results in a mutated geneproduct can have a sequence identity of 70%, 75%, 80%, 85%, 90%, 95%, orgreater to the corresponding non-mutated nucleotide sequence. In someembodiments, a mutated gene which results in a mutated gene product canhave a sequence identity of 96%, 97%, 98%, 99%, or greater to thecorresponding non-mutated nucleotide sequence. In some embodiments ofthe invention, the mutated gene is a mutated human gamma-globin gene. Insome embodiments, the mutated human gamma-globin gene encodes a proteincomprising SEQ ID NO:1.

In some embodiments, the mutated human gamma-globin gene is used togenetically correct sickle cell anemia or β-thalassemia or reducesymptoms thereof, including the steps of identifying a subject in needof treatment for sickle cell anemia or β-thalassemia; transfectingautologous hematopoietic stem cells (HSCs) with a modified lentiviruscomprising the mutated human gamma-globin gene; and transplanting thetransfected HSCs into the subject.

In some embodiments, post-transplantation fetal hemoglobin exceeds atleast 20%; F cells constitute at least ⅔ of the circulating red bloodcells; fetal hemoglobin per F cells account for at least ⅓ of totalhemoglobin in sickle red blood cells; and at least 20% gene-modifiedHSCs re-populate bone marrow of the subject. In some embodiments,post-transplantation fetal hemoglobin exceeds 25%, 30%, 35%, 40%, 45%,50%, or greater. In some embodiments, post-transplantation fetalhemoglobin exceeds 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orgreater. In some embodiments, F cells constitute at least 70%, 75%, 80%,85%, 90%, 95%, or greater of the circulating red blood cells. In someembodiments, fetal hemoglobin per F cells account for at least 1/3 oftotal hemoglobin in sickle red blood cells. In some embodiments, fetalhemoglobin per F cells account for at least 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or greater of total hemoglobin insickle red blood cells. In some embodiments, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greatergene-modified HSCs re-populate bone marrow of the subject.

Essential Cis Elements and the Optimization of Vector Design

As described herein, experimentation was conducted to determine whetherlentivirus non-coding cis-sequences played a specific role in the RNAexport, packaging or expression of β-globin. The vector life-cycle wasstudied in self-inactivating (SIN)-lentiviruses, carrying the β-globingene and locus control region (BG), or GFP cDNA. Systematic analysisstarted with a completely ‘gutted’ minimal SIN-lentivirus carrying onlythe packaging region; and SIN-lentiviruses containing increasing HIVcis-elements, along with a SIN-gamma-retrovirus in order to identifyoptimal cis-elements to include in the SIN-LV backbone. To clone thesSIN-GFP vector, the 3′LTR of a standard SIN-LV backbone previouslyused, as described herein, was modified to improve transcripttermination. Specifically, β-growth hormone polyadenylation signal wasadded downstream of the 3′LTR and a USE sequence derived from SV40 latepolyadenylation signal was added in the U3 deletion.

As further described herein, SIN-gamma-retrovirus or a gutted/minimalSIN-lentivirus encoding GFP generated high titers and mediated high GFPexpression. However, SIN-gamma-retrovirus or the gutted SIN-lentivirusencoding either BG or a similar sized large transgene had barelydetectable titers compared to the SIN-lentivirus carrying cis-elements.Systematic addition of cis-elements demonstrated that Rev/RRE was mostessential, followed by gag and env splice acceptor sequences, forefficient assembly/packaging of lentivirus particles, not mRNA export.However, these HIV cis-sequences were dispensable for smallertransgenes. These studies identify key lentivirus cis-elements and therole they play in vectors carrying large inserts, and have importantimplications for gene therapy.

In one embodiment, the present invention provides a method of increasingtiter of a modified SIN lentiviral expression vector compared to astandard SIN lentiviral expression vector. In another embodiment, theSIN lentiviral expression vector is modified by inserting a heterologouspolyadenylation (polyA) signal sequence downstream from a viral 3′ longterminal repeat sequence in a standard SIN lentiviral vector backbone.In another embodiment, the polyA signal is the bovine growth hormonepolyA signal sequence. In another embodiment, the SIN lentiviral vectoris modified by inserting one or more of an upstream polyA-enhancersequence (USE sequence) into a 3′LTR of a standard SIN lentiviral vectorbackbone. In another embodiment, the USE sequence is derived from theSV40 late polyA signal. In another embodiment, 2-3 copies of the USEsequence are inserted into a 3′LTR of a standard SIN lentiviral vectorbackbone. In another embodiment, 2-10 copies of the USE sequence areinserted into a 3′LTR of a standard SIN lentiviral vector backbone. Inanother embodiment, 3-5 copies of the USE sequence are inserted into a3′LTR of a standard SIN lentiviral vector backbone. In anotherembodiment, one or more copies of the USE sequence is inserted into theU3 region. In another embodiment, the β-growth hormone polyA signal andone or more copies of the USE sequence derived from the SV40 late polyAsignal are both incorporated into the expression vector. In anotherembodiment, the expression vector contains a gene of interest (GOI). Inanother embodiment, the gene is operably linked to a promoter. Inanother embodiment, the promoter is a lineage-specific promoter. Inanother embodiment, the promoter is an erythroid specific promoter. Inanother embodiment, of the GOI is β-globin. In another embodiment, theGOI is gamma-globin. In another embodiment of the invention thegamma-globin gene is under the control of β-globin regulatory elements.In another embodiment, the vector is used to treat sickle cell anemiavia gene therapy. In another embodiment, the vector is used inconjunction with reduced intensity conditioning to treat sickle cellanemia. In another embodiment, the SIN lentivirus comprises a bovine,equine, feline, ovine/caprine or primate derived variety of lentivirus.In another embodiment, the SIN lentivirus is an HIV derived SINlentivirus. In another embodiment the modified SIN lentiviral vector isintroduced into a eukaryotic cell by transfection.

In one embodiment, the present invention provides a method of designinga gutted/minimal, and thus less recombinigenic and safer SIN lentiviralvector for the expression of small therapeutic transgenes that do notrequire extensive Cis elements for efficiency. In another embodiment thesmall therapeutic transgenes are equal in size or smaller than greenfluorescent protein (GFP). In another embodiment the small therapeutictransgenes are smaller than human β-globin.

Chromatin Insulator Elements

As described herein, chromatin insulator elements prevent the spread ofheterochromatin and silencing of genes, reduce chromatin positioneffects and have enhancer blocking activity. These properties aredesirable for consistent predictable expression and safe transgenedelivery with randomly integrating vectors. Overcoming chromatinposition effects can reduce the number of copies required for atherapeutic effect and reduce the risk of genotoxicity of vectors.Vector genotoxicity has become an area of intense study since theoccurrence of gene therapy related leukemia in patients in the X-SCIDtrials. Gamma-retroviral vectors and lentiviral vectors have beenmodified to a self-inactivating (SIN) design to delete ubiquitouslyactive enhancers in the U3 region of the long terminal repeats (LTR). A1.2 Kb DNAse hypersensitive site-4 (cHS4) from the chicken p-globinlocus has been inserted in the 3′LTR to allow its duplication into the5′LTR in gamma-retrovirus and lentivirus vectors. Insulated vectors havereduced chromatin position effects and, provide consistent, andtherefore improved overall expression. A side-by-side comparison of cHS4insulated and uninsulated lentivirus vectors carrying hβ-globin and thelocus control region was performed, and resulted in the discovery thatinsulated vectors showed consistent, predictable expression, regardlessof integration site in the differentiated progeny of hematopoietic stemcells, resulting in a 2-4 fold higher overall expression. Recentevidence also suggests that cHS4 insulated lentivirus vectors may reducethe risk of insertional activation of cellular oncogenes. Despite thebeneficial effects of insulated vectors, they also lead to a significantreduction in titers with insertion of the full-length 1.2 Kb cHS4insulator element in the 3′LTR of lentivirus vectors. There are similarreports of lowering of viral titers or unstable transmission withgamma-retrovirus vectors containing insertions in the 3′ LTR. Thisreduction in titers becomes practically limiting for scale up of vectorproduction for clinical trials, especially with vectors carryingrelatively large expression cassettes, such as the human β-globin gene(hβ) and locus control region (LCR), that have moderate titers evenwithout insulator elements.

The effects of insertions of exogenous fragments into the LTR on virallife cycle have not been addressed. The mechanism by which insertion ofcHS4, or other inserts in the viral 3′LTR lower titers of lentiviralvectors was therefore studied. Large LTR inserts lower titers via apost-entry restriction in reverse transcription, and increasedhomologous recombination in the LTRs of viral cDNA, thus reducing theamount of virus DNA available for integration. These results haveimportant implications for vector design for clinical gene therapy.Studies on the chicken hypersensitive site-4 (cHS4) element, aprototypic insulator, have identified CTCF and USF-1/2 motifs in theproximal 250 bp of cHS4, termed the “core”, which provide enhancerblocking activity and reduce position effects. However, the core alonedoes not insulate viral vectors effectively. While the full-length cHS4has excellent insulating properties, its large size severely compromisesvector titers. A structure-function analysis of cHS4 flankinglentivirus-vectors was performed and transgene expression in the clonalprogeny of hematopoietic stem cells and epigenetic changes in cHS4 andthe transgene promoter were analyzed.

As further described herein, the core only reduced the clonalvariegation in expression. Unique insulator activity resided in thedistal 400 bp cHS4 sequences, which when combined with the core,restored full insulator activity and open chromatin marks over thetransgene promoter and the insulator. These data consolidate the knowninsulating activity of the canonical 5′ core with a novel 3′ 400 bpelement with properties similar to the core. Together, they haveexcellent insulating properties and viral titers. This data hasimportant implications with respect to understanding the molecular basisof insulator function and design of gene therapy vectors.

In one embodiment, the present invention provides a method of increasingthe titer of lentiviral vectors by incorporating one or morereduced-length chromatin insulators containing functional portions of afull-length chromatin insulator. In another embodiment, the functionalportions are derived from a single type of full length chromatininsulator. In another embodiment, the reduced-length functionalinsulator comprises functional portions of two or more separatevarieties of chromatin insulators. In another embodiment, the functionalreduced-length chromatin insulator is derived from a chickenhypersensitive site-4 (cHS4) element. In another embodiment, thefunctional reduced-length insulator is a cHS4-derived insulator of 650base pairs or less. In another embodiment, one or more reduced-lengthcHS4-derived insulators is combined with other modifications to a SINlentivirus expression vector in order to increase titer and improvestability of transgene expression. In another embodiment, one or morereduced-length cHS4-derived insulators is added to a vector containing aheterologous polyadenylation (polyA) signal sequence downstream from aviral 3′LTR and a USE sequence in the U3 deletion.

Sickle Cell Disease

Sickle cell disease (SCD) affects the β-globin gene and is one of themost common genetic defects, resulting in the production of a defectivesickle globin (HbS, comprised of two normal α globin and two β^(sickle)globin molecules, denoted as α₂β^(S) ₂). HbS polymerizes upondeoxygenation and changes the shape of discoid red blood cells (RBCs) tobizarre sickle/hook shapes. Sickled RBCs clog the microvasculature,causing painful acute organ ischemic events and chronic organ damagethat foreshortens the life span of SCD patients to 45 years. Thisdisease affects over 110,000 Americans, with 1000 newborns with SCD bornevery year and nearly 1000 babies born with this disease annually inAfrica.

Therapeutic options for SCD are extremely limited and involve a bonemarrow hematopoietic stem cell transplant (HCT). HCT is available onlyto 10-15% of patients with matched normal sibling donors and is oftenassociated with serious immune side effects. Fetal hemoglobin (HbF,comprised of α and γ globins, α₂γ₂) is produced during the fetal lifeand the first 6-9 months of age and has strong anti-sickling propertiesand protects the infant from sickling in the first year of life. Indeed,individuals with hereditary persistence of HbF that have SCD areasymptomatic. Hydroxyurea, a chemotherapeutic drug that increases HbF,is FDA-approved for ameliorating symptoms of SCD. However, hydroxyureadoes not work for all patients, and due to daily life-long intake, isassociated with poor compliance. Hence, better therapeutic options areneeded for SCD.

Genetic correction of autologous bone marrow stem cells (hematopoieticstem cells) with a lentivirus vector encoding the γ-globin gene would beable to permanently result in production of the anti-sickling HbF,thereby preventing RBC sickling. This method has advantages overcurrently available therapies, including its availability to allpatients, particularly those who do not have a matched sibling donor,and the fact that it would be a one-time treatment, resulting inlifelong correction and devoid of any immune side effects. An effectivegene therapy approach will revolutionize the way SCD is treated andimprove the outcomes of patients with this devastating disorder.

Determining Critical Parameters of Disease Correction—Sickle Cell Anemia

As disclosed herein, lentiviral delivery of human γ-globin underβ-globin regulatory control elements in HSCs results in sufficientpostnatal HbF expression to correct SCA in mice. The amount of HbF andtransduced HSCs was then de-scaled, using reduced-intensity conditioningand varying multiplicity of infection (MOI), to assess criticalparameters needed for correction. A systematic quantification offunctional and hematologic RBC indices, organ pathology, and life spanwere critical to determine the minimal amount of HbF, F cells, HbF/Fcell, and gene-modified HSCs required for reversing the sicklephenotype.

As further disclosed herein, amelioration of disease occurred when HbFexceeded 10%, F cells constituted two-thirds of the circulating RBCs,and HbF/F cell was one-third of the total hemoglobin in RBCs; and whenapproximately 20% sG^(b)G modified HSCs repopulated the marrow. Geneticcorrection was sustained in primary or secondary transplant recipientsfollowed long-term. The present study describes a method of determiningminimum HSC chimerism for correction of a hematopoietic disease in an invivo model, which would contribute to design of cell dose andconditioning regimens to achieve equivalent genetically corrected HSCsin human clinical trials. Moreover, this study addresses the gene dosageand the gene-modified hematopoietic stem cell dosage required forcorrection of a genetic defect.

In one embodiment, the present invention provides a method ofdetermining minimum HSC chimerism for correction of a hematopoeiticdisease in an in vivo model. In another embodiment, reduced intensityconditioning prior to transplantation is used as a method of varying HSCchimerism. In another embodiment, the proportion of transduced HSCs andvector copy/cell is varied by transducing the cells at a range of MOI(30-100). In another embodiment, the MOI is 20-120. In anotherembodiment, the minimum determined chimerism and gene dosage can be usedto design cell dose and conditioning regimens to achieve equivalentgenetically corrected HSCs in human clinical trials. In anotherembodiment, reduced intensity conditioning is used prior totransplantation in a clinical setting to reduce transplantation-relatedmorbidity. In another embodiment, the hematopoeitic disease is sicklecell anemia. In another embodiment, the hematopoeitic disease isβ-thalassemia.

Gene Therapy for Sickle Cell Disease Via Mutant Gamma Globin

As disclosed herein, an improved mutant γ-globin gene has beenengineered from a lentivirus vector, sG^(b)G^(M). This vector has ahigher tendency to form HbF and improved anti-sickling properties,resulting in superior correction of SCD in stringent homozygous SCDmouse models. The engineered γ-globin gene has an increased affinity tobind a-globin without altering its function, thereby greatly improvingthe efficiency of HbF formation in RBCs and resulting in a far moreefficient anti-sickling effect that will correct the SCD phenotype.

As further disclosed herein, the engineered sG^(b)G^(M) vector has atwo-fold higher tendency to form HbF than the native γ-globin gene fromthe sG^(b)G vector and readily corrects the UAB sickle mice efficiently.Both vectors correct SCD in Berkeley sickle mice. Thus, the sG^(b)G^(M)vector provides twice the amount of HbF per vector copy in sickle miceas compared to the sGbG vector. In addition to providing an increasedamount of HbF, the mutant HbF produced from the sG^(b)G^(M) vector alsoconfers sickle RBCs with much longer lifespans as compared to naturalHbF, due to reduced sickling. Accordingly, this vector can efficientlycorrect SCD in human patients.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Lentivirus Cis Elements Required for Efficient Packaging ofLarge Transgenes Cassettes Like β-Globin

This study investigated whether lentivirus non-coding cis-sequencesplayed a specific role in the RNA export, packaging or expression ofβ-globin. The vector life-cycle was studied in self-inactivating(SIN)-lentiviruses, carrying the β-globin gene and locus control region(BG), or GFP cDNA. Systematic analysis started with a completely‘gutted’ minimal SIN-lentivirus carrying only the packaging region; andSIN-lentiviruses containing increasing HIV cis-elements, along with aSIN-gamma-retrovirus. It was discovered that (i) SIN-gamma-retrovirus ora gutted/minimal SIN-lentivirus encoding GFP generated high titers andmediated high GFP expression. (ii) However, SIN-gamma-retrovirus or thegutted SIN-lentivirus encoding either BG or a similar sized largetransgene had barely detectable titers compared to the SIN-lentiviruscarrying cis-elements. (iii) Systematic addition of cis-elementsdemonstrated that Rev/RRE was most essential, followed by gag and envsplice acceptor sequences, for efficient assembly/packaging oflentivirus particles, not mRNA export. However, these HIV cis-sequenceswere dispensable for smaller transgenes. These studies identify keylentivirus cis-elements and the role they play in vectors carrying largeinserts, and have important implications for gene therapy.

Example 2 BG Expression from Gutted SIN-γRV

It has been postulated that γRV are unable to successfully expresshβ-globin due to transcriptional interference between the strong γRV LTRpromoter/enhancer elements and the internal LCR enhancer. SRS11.SF is aSIN-γRV that encodes the GFP cDNA under control of an internal SpleenFocus-Forming Virus (SFFV) promoter/enhancer. The SFFV-GFP in SRS11.SFwas replaced with BG, an expression cassette that was successfullyutilized in a standard SIN-LV to achieve therapeutic human β-globinexpression in thalassemia, to generate SRS11.BG. The rationale for usingSRS.11, despite the notoriety of β-globin γRV was: (i) it contains theminimal packaging region (ψ), lacks gag sequences and can carry a largervector payload, yet retains extremely high titers; (ii) it carries alarge 400 bp U3 deletion of the 3′LTR, comparable to the deletion inSIN-LV. (iii) Large LCR elements have never been tested in γRV due torestrictions on vector payload.

Infectious titers and expression of SRS11.BG and SRS11.SF γRV vectorswere compared on the murine erythroleukemia (MEL) cell line. Humanp-globin protein expression was almost undetectable fromSRS11.BG-transduced MEL cells, in contrast to the high expression of GFPin cells transduced with SRS.11 SF. The unconcentrated viral titers ofSRS11 BG versus SRS11.SF vector were 6.8±5×10³ IU/mL versus 4±0.2×10⁶IU/mL. Viral RNA (vRNA) transcripts were barely detectable in 293T cellswith the SRS11.BG via northern blot analysis (data not shown).Therefore, production of BG vRNA and viral particles from γRV, eventhose optimized for a SIN design and high vector payload was severelyimpaired.

Example 3 Expression of Large/SmalI Transgenes from Standard orGutted/Minimal LV

In contrast to the SIN-γRV used herein, the “standard” SIN-LV commonlyused retains relatively large portions of viral sequences amounting toabout 20-25% of the HIV genome. These cis elements are: the LTR (634 bpfor wt HIV LTR or 235 bp for SIN-LV LTR), the packaging signal ψ(150bp), 5′ portion of the gag gene (300 or 600 bp), env sequences includingthe rev response element (RRE, 840 bp) and the central flap/polypurinetract (cPPT) from the pol gene (120 bp).

To examine the requirement of cis-sequences for GFP versus BG, theCMV-GFP cassette was cloned in a) the “standard” SIN-LV containing cissequences listed above (sSIN-GFP), and b) a ‘gutted’ minimal SIN-LVwhere the gag, RRE and the rest of the env sequences were deleted andonly the ψ region was retained (dsSIN-GFP; FIG. 1A). The titers of theminimal dsSIN-GFP LV were only 2-times lower than the titers of the“standard” LV sSIN-GFP FIG. 1B; p<0.01; n=3. In sharp contrast to theGFP vectors, the difference in titers of the analogous standard andgutted BG SIN-LV, sBG-6 and sBG-1 vectors was 1100-fold p<0.01; n=4;(FIG. 1B). Clearly, the LV non-coding sequences are necessary either forproduction of LV particles and/or for β-globin expression; and thesesequences have a pronounced effect on infectious titers of LV encodingthe β-globin gene, but not those encoding GFP. Next, vectors wereconstructed with a similar size transgene cassette, CMV-FANCA-IRES-GFP(FIG) as sBG (FIG. 1A) in the “standard” (sFIG) or the gutted (dsFIG)SIN LV. The same dependence of FIG on LV cis sequences: titers of dsFIGvector were three orders of magnitude lower than those of sFIG wereobserved (FIG. 1B). Therefore LV cis elements are dispensable for smallinserts, but necessary for high titers of large inserts.

Example 4 LV Constructs Designed to Study the Role of Cis-Sequences

To study which particular LV cis sequences were important for thiseffect, and what step of the vector life cycle they affected, a seriesof ten SIN-LV vectors were cloned; all of them carrying the BG cassettebut carrying different lentiviral non-coding cis elements (FIG. 2). Therationale for studying specific env (RRE and SA) and gag sequences inthe context of BG was: (i) The RRE element in the env fragment in a“standard” LV facilitate transport of unspliced/singly splicedtranscripts from the nucleus following binding with the Rev protein.(ii) The env splice sites play a fundamental role in the stability ofvRNA and its availability for packaging, and absence of known downstreamsplice acceptor (SA) sequences results in cis-acting repressor sequence(CRS) activity, which hinders cytoplasmic accumulation of HIV-1 RNA.(iii) A portion of the gag gene is retained in vectors to help vRNApackaging. Gag sequences promote folding of the RNA secondary structureof the packaging signal, facilitate the interaction of vRNA with Gagproteins during particle formation, and are important for thedimerization of the vRNA. Sequences mapped to the 5′ splice donor siteand the first 360 bp of the gag gene direct unspliced and singly splicedviral mRNA to specific subnuclear compartments from where it is exportedwith the help of Rev/RRE.

The first vector (sBG-1) maintained only the packaging signal(containing the 5′ splicing donor site) and the cPPT/flap (FIG. 2).Starting from this vector, the RRE, the rest of the env fragmentcontaining the SA, and two different size gag fragments (360 bp and 630bp) were sequentially cloned into sBG-2, sBG-3, sBG-5 and sBG-6. Toverify the activity of the splicing acceptor (SA) the sequence in theenv fragment was mutated by PCR site-specific mutagenesis (sBG-4). Inthe last four vectors, the entire env fragment including the RRE wasfirst removed, leaving only the long and short version of the gagfragments (sBG-9, sBG-10); or additionally added RRE (sBG-7, sBG-8)downstream of the long and short gag fragments.

Example 5 Viral Titers with Inclusion of Different HIV Cis Sequences

The vectors without the RRE element (sBG-1, sBG-9 and sBG-10) had aconcentrated titer ranging from 5.5±2.1×10⁵ IU/mL to 1.7±1.4×10⁶ IU/mL,which was 2-3 orders of magnitude lower than vectors that carry the RREsequence (sBG-2 to sBG-8; p<0.01). Indeed when only the RRE sequence wasadded to sBG-1 to generate sBG-2, the titer increased by more than a100-fold (5.5±2.1×10⁵ IU/mL versus 8.7±6.5×10⁷ IU/mL; p<0.01; FIG. 3).

Addition of the env fragment containing the SA site increased vectortiters 3-5 fold: 2.9±0.9×10⁸ IU/mL for sBG-3 versus 8.7±0.7×10⁷ IU/mLfor sBG-2 (p<0.01). This effect was specific to the SA, since titers ofsBG-4 vector, which contains the env sequence with a mutated SA were1.1±0.61×10⁸ IU/mL, and were similar to that of sBG-2 carrying only theRRE (sBG4 vs. sBG-3 p<0.01). The addition of a long and short fragmentof gag to env (RRE and SA) containing vectors sBG-5 and sBG-6,respectively, showed a further increase in titers by ˜4-5 fold, withtiters from sBG-6 reaching 6.3×10⁸ IU/mL (sBG-4 vs. sBG-5 and sBG-6p<0.01). The data suggested that the longer portion of gag was notnecessary for high BG titers. However, titers of vectors carrying onlythe short/long gag fragments, without the RRE and env SA were low (sBG-9and sBG-10), as compared to those containing the RRE as well (sBG-7 andsBG-8; p<0.01). Titers of sBG-7, 8, 9, and 10 ranged from 9.4±4.7×10⁵IU/mL to 1.4±0.4×10⁸ IU/mL. Titers improved further by 3-5 fold with theinclusion of env SA. Thus, the gag fragment alone, or the combinationgag/RRE was not sufficient to confer optimal titers to BG vectors,suggesting HIV-1 cis sequences acted cooperatively.

To study whether the strong effect of the RRE on viral titers wasRev-dependent, the sBG-6 vector was packaged with and without Rev. Inthese experiments, the packaging system was changed from 3-plasmid to a4-plasmid system, wherein Rev and Gag-Pol were provided from differentplasmids. The titers of sBG-6 were approximately 400-fold higher withRev (3.8±0.3×10⁷ IU/mL) than without the Rev protein (9.4x±5.8×10⁴IU/ml; p<0.01), showing that interaction of Rev with RRE was necessaryfor high titers.

Taken together, these data indicate that HIV-1 Rev/RRE, gag and env SAwere critical for high titers of LV carrying a large cargo such as BG orFIG, although they are dispensable for small GFP based cassettes.

Example 6 Role of LV Cis-Elements in the Vector Life Cycle

In order to assess the role of LV cis-elements in proviral stability andexpression a genomic Southern blot analysis on transduced MEL cells wasperformed.

Surprisingly, given previous difficulties with genomic rearrangements ofhβ-globin-containing γRV, only one proviral band of the expected sizewas detected in most of the LV FIG. 4A. Some low titer vectors wereundetectable at the level of sensitivity of a Southern blot. Subsequentnorthern blot analysis in packaging cells confirmed that the expectedfull-length vRNA transcripts were generated from all LV (FIG. 5A),confirming that LV carrying the large BG cassette do not requirecis-sequences for stable transmission.

In order to determine whether LV cis-sequences affected the level ofexpression of integrated BG proviruses, MEL cells were transduced withvectors sBG-1 through sBG-10 at a range of multiplicity of infection.Mean fluorescence intensity (MFI) was compared in MEL cell pools with asimilar percentage of hβ-globin expressing cells (15-20%), except invectors with low titers, where only a small percentage of gene transfercould be achieved. The MFI of the transduced MEL cell population wascomparable among all the vectors (ranging from 62 to 110 arbitraryunits), including that of the low titer vectors (FIG. 4B). Thus, LVcis-elements did not play a major role in regulating the expression ofBG.

In order to determine the role of RRE, gag and env SA in vRNA productionand cytoplasmic export the steps of vector life cycle that could impairgeneration of full-length vRNA in the packaging cells, its subsequentcytoplasmic export, assembly and packaging into vector particles wasstudied.

Total, cytoplasmic and nuclear RNA was fractionated from 293T packagingcells transfected with sBG-1 through sBG-10. FIG. 5A shows a northernblot analysis on total RNA probed with hβ-globin probe. Correctly sizebands of intact vRNA from all the vectors, including the vectors withoutthe RRE were determined (sBG-1, sBG-9 and SBG-10). The spliced andunspliced vRNA transcripts were only present for the vectors sBG-3,sBG-5 and sBG-6, since these vectors carry the env SA site. Thus, noappreciable aberrant splicing occurred in any of the LV backbones,confirming lack of recombination of the hβ-globin gene and LCR elements,and contrasting results reported with γRV.

Significantly, all vectors with very low titers, including sBG-1, sBG-9and sBG-10 that do not contain RRE, produced vRNA in quantities thatwere comparable to, or higher than the highest titer vectors (sBG-5 andsBG-6). Since this finding was unexpected, the northern blot wasrepeated in a separate experiment, with fractionation of total andcytoplasmic RNA, with identical results.

Rev/RRE has been best characterized for export of full-length vRNA tothe cytoplasm. Therefore, the next step was to determine if RREcontributed to high titers via vRNA export. Northern blot analysisshowed similar amounts of vRNA in the cytoplasm of analogous vectorswithout or with RRE (sBG-1 versus sBG-2, sBG10 versus sBG-8, and sBG-9versus sBG-7; FIG. 5B). The ratio of cytoplasmic RNA to total RNA innorthern blots from two separate experiments is shown in 6E. Thecytoplasmic vRNA transcripts were only 2-fold higher in sBG-2, whencompared to sBG-1. The converse was seen with sBG-10 and sBG-9 vectors,where cytoplasmic vRNA transcripts were ˜2-fold higher than analogousvectors sBG-8 and sBG-7, which contained the RRE. Since the differencein titers between vectors with and without the RRE was 2-3 orders ofmagnitude, RRE likely played a minimal role in increasing nuclear exportof vRNA transcripts via these vectors.

Example 7 LV Cis-Elements, Including RRE Improve Packaging

The effect of cis sequences on the packaging efficiency was nextdetermined by analyzing vRNA, p24 levels and viral associated reversetranscriptase (RT) in purified virus particles from all ten vectorsprocessed identically. FIG. 6A shows a representative dot blot analysisof sBG1 through sBG-10 LV. The amount of vRNA detected is proportionalto the vector titer for most of the vectors, as determined byphospho-imager analysis, indicating a block in packaging efficiency invectors lacking cis-sequences (FIG. 6B-C).

There were some exceptions that suggested cis-sequences may have someeffect on steps following target cell entry: RNA in 293T cells and theinfectious titers of sBG-2 and sBG-4 were comparable, although sBG-4vRNA was 4-times higher. It seems that sBG-4 vRNA, even when packagedmore efficiently, may not be stable post-target cell entry due to theabsence of env SA, which is known to stabilize RNA. sBG-6 and sBG-7 hadthe same amount of vRNA but the titer of sBG-7 was 4-5 times lower; hereagain sBG-7 did not have the env SA. sBG-5, containing the inhibitoryregion of gag, had higher vRNA, but lower titers.

Overall, the amount of BG vRNA packaged in viral particles correlatedwith the transduction/infectious titers in target cells, despite highlevels of mRNA produced in packaging cells with all 10 vectors. The p24activity was similar in all the concentrated virus preparations (FIG.6D), suggesting that viral like particles (containing no vRNA) wereformed efficiently with all vectors.

Example 8 γRV and LV Size, Payload and Titer

Titers of the standard LV carrying BG are low to begin with, and requireextensive concentration. However, the titers fall precipitously (bythree orders of magnitude) with the removal of LV cis-elements. Perhapsthese LV sequences protect large vRNA from degradation in packagingcells while promoting assembly, while the short GFP vRNA getsefficiently packaged without such requirements. The low titer of the‘gutted’ BG LV are not from anti-sense RNA arising from the β-globingene promoter inserted in the reverse orientation with respect to the5′LTR vRNA transcript in 293T cells. There was no antisense transcriptin the northern blot with any of the vectors. Besides, β-globintranscripts are erythroid-specific, and are not produced in 293T cells.Furthermore, the FIG cassette that was similar in size to BG, but insense orientation also had the same effect on titers as BG.

Example 9 Cis Elements and Vector Life-Cycle

Several unique, rather unexpected results emerged from this study: (i)in packaging cells, large amounts of transcripts were produced with allBG LV in contrast to barely detectable RNA with BG γRV. One possibilityis that LV minimal sequences (R, U5 and Ψ regions) confer stability toBG vRNA in specific sub-cellular compartments. Therefore, high amountsof vRNA are seen in 293T cells even from the gutted LV, an essentialdifference from the BG γRV. (ii) BG vRNA was of the expected size andefficiently exported into the cytoplasm even in the absence of Rev/RRE,contradicting the belief that the success of ‘ globin genes’ in LV issecondary to the archetypal functions of RRE of preventing splicing andvRNA export. (iv) vRNA was efficiently packaged into virions when thegutted LV encoded a small transgene such as GFP. This data confirms LVcis-sequences, other than the minimal packaging sequence, aredispensable for small transgenes.

Example 10 Role of RRE in Packaging

Rev/RRE interaction was most critical for packaging and high titer virusproduction, while the well-established function of Rev/RRE in the exportof the genomic vRNA and suppression of spliced message was not prominentin BG LV. In wild type HIV virus, the presence of Rev/RRE is requiredalong the entire mRNA transport and utilization pathway for thestabilization, correct subcellular localization, and efficienttranslation of RRE-containing mRNA. The data presented here confirms andextends a recent study that shows that RRE had a minor effect oncytoplasmic vRNA levels, but reduced viral titers approximately100-fold. It further shows that Rev/RRE requirement is specific forlarge transgenes, but dispensable for small expression cassettes. Unlikea previous report in the literature, the present research did not see arole of RRE in vRNA stabilization, since equal or higher amounts of vRNAwas seen with vectors without RRE. The likely mechanism is the capacityof RRE to be involved in viral assembly and packaging.

Example 11 Role of Env SA and Gag Sequences

Presence of the env SA has been shown to stabilize the viral genome,resulting in a higher virus production. Presence of SA may alsostabilize the vRNA at a post-entry level, since some vectors without theenv SA, when compared to analogous vectors with the env SA had the samev-RNA but had lower transduction/titer in target cells. The gagsequence, with a start codon mutation to prevent the translation of thegag protein, helps the production of LV during viral packaging. In thisstudy it was determined that this requirement was specific to largetransgene cassettes. It was also demonstrated that removal of aninhibitory sequence present between 414 bp and 631 bp of the gag genethat has been previously shown to decrease the stability ofgag-containing RNAs, increased titers by 3.5-fold.

In conclusion, this research describes the steps in the viral life-cycleaffected by the non-coding cis-sequences when LV encodes large transgenecassettes; and their dispensability for smaller transgenes such as GFP.These results provide new insight in the design of LV vectors.Gutted/minimal LV could be designed for small therapeutic transgenes,which would be less recombinogenic and safer in gene therapyapplications.

Example 12 Viral Vector Design

LV: To clone the sSIN-GFP vector, the 3′LTR of a standard SIN-LVbackbone previously used was modified to improve transcript termination:β-growth hormone polyadenylation signal was added downstream the 3′LTRand a USE sequence derived from SV40 late polyadenylation signal wasadded in the U3 deletion. The dsSIN-GFP was obtained by removing theClaI-NruI fragment from the sSIN-GFP plasmid. A multi-cloning site(MCS-ClaI-Eco47III, XhoI, SmaI, SalI, EcoRI:CCATCGATAGCGCTCTCGAGCCCGGGGTCGACGAATTCC) was cloned in the ClaI andEcoRI sites of sSIN. The β-globin-LCR (BG) cassette was cloned inreverse orientation into the XhoI and SmaI sites and this parentconstruct was termed sSIN-BG. sBG-0 was obtained removing the regionbetween Eco47III and NruI, leaving behind only HIV-1 packaging sequence(ψ) following the 5′LTR from sSIN-BG. cPPT was cloned into sBG-0 ClaIsite (sBG-1). PCR fragments for RRE, RRE-env, short gag (360 bp), longgag (630 bp) were cloned in XhoI blunted site, and these vectors weretermed sBG-2, sBG-3, sBG-10, sBG-9, respectively. Primers sequences,where F denotes forward primers and R denotes reverse primers:

RRE_F: ATAAACCCGGGAGCAGTGGGAATA; RRE_R: ACATGATATCGCAAATGAGTTTTCC;ENV_R: ACATGATATCATACCGTCGAGATCC;GAG_F: ACTGCTCTCGAGCAATGGGAAAAAATTCGGT;GAG_1R: ACTGCTCTCGAGGCAGCTTCCTCATTGATG;GAG_2R: ACTGCTCTCGAGATCAGCGGCCGCTTGCTGT.

A frame-shift mutation was inserted in the 5′ sequence of gag in thestart codon to disable the gag start site, using the primer Gag F thatinserts the dinucleotide CA in the gag ATG. Vectors sBG-7 and sBG-8 wereobtained cloning long gag and short gag PCR fragments into XhoI site ofsBG-2. A point mutation to disrupt the SA site in the env sequence wasperformed using MutSA_F (TATCGTTTCGAACCCACCTCC) and MutSA_R(GGAGGTGGGTTCGAAACGATA) primers to generate sBG-4 (the wt SA sequenceCAG inside the Env fragment was mutated into CGA). sBG-5 was obtainedcloning the long gag PCR fragment into the XhoI site of sBG-3. γRV:SRS11.SF γRV plasmid was kindly provided by Drs. Axel Schambach andChristopher Baum, (Hannover, Germany). In SRS11.BG vector, the humanβ-globin-LCR (BG), was cloned in reverse orientation into the PstI siteof SRS11.SF retroviral vector plasmid. All vector cartoons are depictedin FIG. 2.

Example 13 Virus Production

LV was produced by transient co-transfection of 293T cells, aspreviously described using the vector plasmids, the packaging (Δ8.9) andthe envelope (VSV-G) plasmids; virus-containing supernatant wascollected at 60 hours after transfection and concentrated byultracentrifugation. All vectors in an experiment were packagedsimultaneously and the virus was concentrated 1400-fold from all viralsupernatants by ultracentrifugation at 25,000 rpm. Viral titers weredetermined by infecting mouse erythroleukemia (MEL) cells or HT1080cells with serial dilution of concentrated virus, differentiating them,and analyzing them for HbA or GFP expression by fluorescence-activatedcell-sorter (FACS) as previously described. γRV were produced similarlybut not concentrated. All transfections and subsequent titration wereperformed in triplicate. Packaging of vectors, with and without Rev, wasperformed following a similar method, except that the packaging plasmidΔ8.9 was replaced with pMDLg/pRRE and pRSV-Rev. The ratio of vectorplasmid:pMDLg/pRRE:pRSV-Rev:VSV-G was 4:4:3:1.

Example 14 Cell Lines

Murine erythroleukemia cell (MEL) line and 293T cells were maintained inDulbecco modified Eagle Medium (DMEM, Mediatech, Inc., Herndon, Va.)supplemented with 10% heat inactivated fetal bovine serum (FBS) (U.S.Bio-technologies, Inc, Parker Ford, Pa.). MEL cells were induced todifferentiate in DMEM containing 20% FBS and 5 mM N,N′-hexamethylenebisacetamide (Sigma), as previously described in the art.

Example 15 HbA Staining and FACS Analysis

The methodology used to label human β-globin using the anti-human HbAantibody was as previously described. Briefly, cells were fixed in 4%paraformaldehyde for 60 minutes at room temperature, washed once withphosphate-buffered saline (PBS), and the pellet resuspended in 100%methanol for 5 minutes. The fixed cells were then washed with PBS, andnonspecific antibody (Ab) binding was blocked using 5% nonfat dry milkfor 10 minutes at room temperature. Subsequently, cells were washed inPBS, pelleted, and permeabilized. The cells were divided into 2 tubesand stained with either anti-zeta globin-fluorescein isothiocyanate(FITC) Ab (1 μg/10⁶ cells) as a negative control or anti-HbA-FITC Ab(0.1 μg/10⁶ cells) (Perkin Elmer, Waltham, Mass.) for 30 minutes at roomtemperature in the dark. Unbound Ab was removed by a final wash with PBSbefore they were analyzed on FACS Calibur (Becton Dickinson, FranklinLakes, N.J.).

Example 16 Total and Cytoplasmic RNA Northern Blot

293T cells were harvested and washed in PBS 72 hours after transfection.Isolation of nuclear and cytoplasmic RNA is obtained with a 7 minutesincubation on ice with NEB buffer (10 Mm Tris-HCl pH 7.4; 10 mM NaCl, 3mM MgCl2; 5% IGEPAL). After centrifugation RNA-STAT (Tel-Test, INC,Texas) was added to the supernatant that contains cytoplasmic RNA, andproceeded with RNA extraction following manufacturer's instructions.Total RNA was extracted from 293T cells using RNA-STAT. Northern Blotwas then performed according to standard protocol. The blot washybridized with a ³²P labeled β-globin probe. To normalize the loadingof the RNA, membranes were then stripped and re-probed with a ³²Plabeled 18S probe. To test the purity of cytoplasmic RNA membranes werestripped and re-probed with a ³²P labeled probe specific for GAPDHintron probe that detected no intronic transcript in the cytoplasmicpreparation.

Example 17 Genomic Southern Blot

Genomic DNA was performed on DNA isolated from transduced MEL cells and10 μg of genomic DNA was digested with AflII enzyme and Southern Blotperformed according to standard protocol. The blot was hybridized with aHS2 fragment of the β-globin LCR probe. RNA dot blot vRNA was extractedfrom same volumes of concentrated viruses using the QIAamp vRNA Mini Kit(Qiagen) following the manufacturer's instructions. Briefly the viruswas lysed under highly denaturing conditions and then bound to asilica-gel-based membrane. Two washing steps efficiently washed awaycontaminants and vRNA was eluted in 30 μl of DEPC-water. After elutionvRNA was treated for 20 min at room temperature with DNAse I,amplification grade DNase I (Invitrogen, Carlsbad, Calif.) wasinactivated by incubating the sample at 65°. vRNA was then denatured in3 vol of denaturation buffer (65% formamide, 8% formaldehyde, MOPS 1×)for 15 min at 65°. After denaturation 2 vol. of ice-cold 20×SSC wereadded and the RNA was bound to a nylon membrane by aspiration through adot-blot apparatus. The blot was hybridized with a ³²P labeled β-globinspecific probe and an X-ray film was exposed overnight.

Example 18 Chromatin Insulators—Generally

Chromatin insulators separate active transcriptional domains and blockthe spread of heterochromatin in the genome. Studies on the chickenhypersensitive site-4 (cHS4) element, a prototypic insulator, haveidentified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4,termed the “core”, which provide enhancer blocking activity and reduceposition effects. However, the core alone does not insulate viralvectors effectively. The full-length cHS4 has excellent insulatingproperties, but its large size severely compromises vector titers. Astructure-function analysis of cHS4 flanking lentivirus-vectors wasperformed and transgene expression in the clonal progeny ofhematopoietic stem cells and epigenetic changes in cHS4 and thetransgene promoter were analyzed. The core only reduced the clonalvariegation in expression. Unique insulator activity resided in thedistal 400 bp cHS4 sequences, which when combined with the core,restored full insulator activity and open chromatin marks over thetransgene promoter and the insulator. These data consolidate the knowninsulating activity of the canonical 5′ core with a novel 3′ 400 bpelement with properties similar to the core. Together, they haveexcellent insulating properties and viral titers. This data hasimportant implications with respect to understanding the molecular basisof insulator function and design of gene therapy vectors.

Example 19 Vector Constructs and Experimental Design

Self-inactivating lentivirus vectors were designed to incorporate eitherthe 5′ 250 bp “core” (sBGC), two tandem repeats of the core (sBG2C), 5′400 bp (sBG400), 5′ 800 bp (sBG800) or the full-length 1.2 Kb cHS4insulator (sBG-I). All vectors carried the human (h)β-globin gene andpromoter and the locus control region enhancer. The different insulatorfragments were cloned in the forward orientation into the U3 region of3′ LTR, so that upon reverse transcription, integrated provirus intarget cells has the insulated 3′ LTR copied to the 5′LTR, and flanksthe hβ-globin expression cassette at both ends. To assess whetherelements outside the 5′ 250 bp core merely provided a spatial scaffold,vectors with inert DNA spacers downstream of the core, sBG400S andsBG800S, were also tested. All vectors were compared to the uninsulatedcontrol, sBG (FIG. 7A).

First, MEL cells were infected with each of the lentivirus vectors andsingle integrant MEL clones were identified (FIG. 7B). All analysis wasperformed only on single-copy MEL clones that carried hβ-globin andverified to have intact insulator sequences by PCR, and subjected toqPCR for vector copy number; hβ-globin expression was analyzed byFACS: 1) the percentage of hβ-globin expressing cells (% hβ+ cells) wasused to determine chromosomal position effects, and 2) the variation ofexpression of hβ-globin expression in cells within a clone, asdetermined by the coefficient of variation (CV), was used to determinethe clonal variegation in expression (FIG. 7C). ChIP analysis wasperformed on the histones over the insulator regions and hβ-globin genepromoter in the different proviruses to study epigenetic modifications.Chromatin position effects of these vectors were confirmed in vivo, inRBC of Hbbth3/+ thalassemia mice transplanted with vector-transducedHSCs 24 weeks after transplant. Secondary transplants were thenperformed and single-integrant CFU-S following transplants were analyzedfor hβ-globin protein and mRNA. In mice, hematological analysis, andHPLC for hβ-globin protein were additionally performed to quantifyexpression.

Example 20 Regions of cHS4 Necessary to Protect from Chromatin PositionEffects

Consistent with previous results, a very high % of hβ+ cells werepresent in the sBG-I single-integrant clones compared to control sBGclones (P<0.01); the % of hβ+ cells in sBGC, sBG2C, sBG400 and sBG800clones were not significantly different from the sBG control clones(FIG. 8A) In order to ensure that the presence of cHS4 in the LTR didnot bias integration, and that the analysis was performed on distinctclones, by LM PCR and integration site sequencing on ten randomlyselected sBG or sBG-I MEL clones. Insertions occurred near/in distinctgenes between uninsulated and insulated clones, with no apparent bias.The presence of the cHS4 core (sBGC), or extended sequences of theinsulator downstream to the core, up to 800 bp, did not increase the %hβ+ cells further; neither did tandem repeats of the core sequence, eventhough the latter has been shown to confer enhancer blocking effect inplasmid-based systems.

Another phenomenon seen with transgene expression is clonal variegation,defined as varying levels of expression in daughter cells with the sameintegration site. A quantitative way to determine clonal variegation isby FACS analysis of transduced clones and calculation of the coefficientof variation (CV) of expression of the transgene around the averageexpression of the transgene in the clone. The CV is a unit-less measureof variability calculated as ratio between sample standard deviation(SD) and the sample average. A high CV was observed in the uninsulatedsBG clones (FIG. 2B). The CV was significantly reduced in all vectorsthat contained the 5′ 250 bp core. These results were confirmed inclones derived from vectors that carried inert DNA spacers downstream ofthe core: sBG400S and sBG800S, showing that the reduction in CV wasspecific to the insulator core, and in contrast to the data on % of hβ+cells, which required the full-length insulator to be present.

It was notable that PCR for insulator sequences showed absence of theinsulator sequences only in sBG2C proviruses, with 6 of 24 clones (25%)MEL clones having both copies of the core deleted from both LTRs. Therewas no observed deletion of the insulator sequences in clones from allother vectors. Southern blot analysis of sBG2C MEL pools confirmeddeletion of one/both copies of the core in the majority of cells.Reverse transcription of repeat sequences, known to result inrecombination events in retroviral vectors likely caused unstabletransmission of the vector with repeat core sequences. This effect ofthe core versus the full-length cHS4 was confirmed in vivo, inthalassemia mice. Peripheral blood RBC were analyzed for hβ-globinexpression 6 months following transplant. FACS analysis in RBC from sBG,sBGC, sBG2C, sBG400 and sBG-I groups of mice (representative plots shownin FIG. 9A) shows that the % hβ+ RBC were significantly higher only inthe sBG-I group of mice, compared to sBG group of mice, like the data inMEL cells; and the CV was significantly lower in all vectors thatcarried the core (P<0.01; FIG. 9B-C). Taken together, this dataindicates that the full-length cHS4 is required to shield againstchromosomal position effects.

Example 21 Chromatin Position Effects in the Clonal Progeny of MurineHSC Following Secondary Transplants

The chromatin position effects were next confirmed in single copysecondary CFU-S. The secondary colony forming units-spleen (CFU-S) assayis considered the most stringent assay that is a ‘gold-standard’ forstudying epigenetic effects of chromatin insulator elements in cellsderived from hematopoietic stem cells. Notably, no transduced CFU-S thatwas positive by PCR for vector-specific sequences that did not expresshβ-globin by FACS were observed, consistent with results reported onlack of transgene silencing with erythroid-specific SIN lentivirusvectors. FACS analysis for (1) % hβ+ cells and (2) TER-119 positiveerythroblasts showed no difference in the percentage of TER-119+ cellsbetween different vector groups (not shown). However, significantlyhigher % of hβ+ cells were only present in secondary CFU-S with thesBG-I vector. Again, the CV was significantly lower in CFU-S transducedwith all the vectors carrying the core, compared to uninsulated sBGtransduced CFU-S (FIG. 3D-E). Real-time RT-PCR analysis on six randomlyselected CFU-S from each group of mice showed that compared to the sBGvector, mRNA expression from the sBG-I CFU-S was approximately 2-foldhigher. However, expression from sBGC, sBG2C and sBG400 transduced CFU-Swas not significantly different from that of sBG CFU-S. Taken together,these data indicate that the 5′ 250 bp core sequences in sBGC, sBG400,sBG400S, sBG800 and sBG800S specifically reduced the clonal variegationof hβ-globin expression. However, the full-length cHS4 element wasrequired for improved probability of expression from differentintegration events.

Example 22 Patterns of Histone Acetylation and Methylation in the CoreRegion and the β-Globin Promoter Region in Insulated Vectors

Next the epigenetic modifications that accompany the specific effectsseen with the various insulator regions were determined by comparing therelative levels of active histone marks acH3, acH4 and H3K4me2 andrepressive histone marksH3K9me3 and H3K27me3 between differentproviruses in MEL clones. ChIP analysis was performed on the cHS4 corein three representative clones that were pooled together for each vector(clones chosen are shown as filled circles in FIG. 8A) bysemi-quantitative PCR (FIG. 10B-C) and real-time PCR) (FIG. 10D-F).Clones carrying the sBG-I vector integrants showed approximately 6-foldenrichment of the active chromatin marks and decreased repressivechromatin marks over the cHS4 “core” fragment, compared to sBGC, sBG400and sBG800, three vectors that carried the “core”.

Histone modifications were analyzed over the hβ-globin promoter in theuninsulated vector (sBG) and all other vectors, which carried the“core”, to assess whether differences in histone patterns over thetransgene promoter in vectors may have contributed to the reduced clonalvariegation. There was a small but significant reduction in repressivechromatin patterns H3K27me3 with sBGC, sBG400 and sBG800 proviruses,compared to the uninsulated sBG provirus (FIG. 10F, right panel).However, with the sBG-I provirus, where maximal insulator activity waspresent, the hβ-globin promoter region had markedly reduced repressivechromatin patterns.

These data show that the “core” sequences and extension of the core upto the 5′ 800 bp of cHS4 reduced activation marks over the transgenepromoter to a small extent. However, a major reduction in repressedhistone modifications over cHS4 and the transgene promoter region onlyoccurred when the distal 3′ 400 bp sequences of cHS4 were present inaddition.

Example 23 Hematological Parameters in Thalassemia Mice Transplantedwith HSCs Transduced with Uninsulated and Insulated Vectors

The anemia, reticulocytosis and other RBC indices were improved evenwith the sBG vector (FIG. 11A), consistent with published reports withuninsulated hβ-globin lentivirus vectors. Hemoglobin ofmock-transplanted mice was 7.7±0.2 gm/dL and the sBG group of mice was10.4±0.7, with 1.2 vector copy per cell. It was noteworthy that thesBG-I group of mice had higher hemoglobin and the lowest reticulocytecount, despite having half the vector copies per cell compared to thesBG group of mice (hemoglobin 11±0.2 gm/dL; 0.6 vector copies per cell).When normalized for transduction efficiency, this amounts to a 5.2 gmincrease in hemoglobin per vector copy in sBG-I mice over mock mice, incontrast to a 2.3 gm increase in hemoglobin per vector copy in the sBGmice. RBC parameters from the experimental mice showed significantimprovement (FIG. 11A; note that these data are not normalized fornumber of vector copies). Improvement in these indices was highest withthe sBG-I mice, albeit not significantly different unless normalized forvector copy.

HPLC analysis for hβ-globin protein in blood confirmed significantlyhigher hβ-globin expression only in the sBG-I mice: 43±3% of the totalhemoglobin in RBC was derived from hβ-globin (hβ2mα2) in sBG-I mice ascompared to 19±6% in the sBG mice, while that in sBGC, sBG400 and sBG2Cgroup of mice was not significantly different from control (FIG. 11B).Human hβ-globin expression and hematological parameters in the sBG2Cgroup of mice were similar those seen in the uninsulated control group.

Example 24 Insulator Activity in the 3′400 cHS4 Region

Since the 5′ 800 bp of cHS4 only reduced the CV, while full insulatoractivity was restored with the full-length 1.2 Kb insulator. A vectorwas generated carrying only the distal/3′ 400 bp region of the cHS4(sBG3′400) derived MEL clones and mice were transplanted withsBG3′400-transduced LSK cells. Note that unlike vectors describedearlier, this vector does not contain the 5′250 bp “core” sequences(FIG. 12A). The sBG3′400 vector had no effect on % of hβ+ cells in MELclones or the % hβ+ RBC in mice (FIG. 6B,D), an effect comparable to sBGclones, or those carrying the 5′ 250 bp “core” (sBGC). However, like allvectors carrying the 5′ core, sBG3′400 significantly reduced the CV ofhβ-globin expression in MEL clones and in RBC (FIG. 12C,E).

The amount of hβ-globin protein in the sBG3′400 mice, determined by HPLCanalysis, was not significantly different from sBG (17.5±3% versus19.5±5.6%), but was at least 2-fold lower than that seen in the sBG-Imice (43±3%; P<0.01) (FIG. 12F). Overall, the 3′ 400 bp of cHS4 hadactivity that was very similar to the 5′ 250 bp core (FIG. 9): itreduced clonal variegation, reflected in a reduced CV of hβ-globinexpression in MEL clones and in RBC, but had no effect on the proportionof hβ-globin expressing red cells. “Core-like” effects of the 3′ 400 bpin individual single copy secondary CFU-S (FIG. 12G-H), were confirmed,with results similar to those with the sBGC vector (FIG. 9D-E). The 3′400 region has no known consensus sequences for CTCF or USF-1, and thisregion has not been previously analyzed. It was noteworthy that neitherthe 5′ core, nor the 3′ 400 bp, when present alone, were able to improvethe probability of expression of integrants/protect from positioneffects.

Example 25 Insulator Activity of the 5′ “Core” Combined with the 3′ 400bp

When the 5′ 250 bp core and the 3′ 400 bp sequences of cHS4 insulator(sBG650 vector; FIG. 13A) were combined, this vector performed similarlyto the sBG-I vector—in MEL clones, in RBCs of transplanted mice and insecondary CFU-S. The proportion of hβ-globin expressing cells in sBG650MEL clones and RBC (FIG. 13B-D) was significantly higher compared to sBGclones (P<0.001), and was similar to sBG-I clones. Likewise, the CV ofthe sBG650 clones was comparable to sBG-I clones (FIG. 13C). Thehβ-globin expression in the RBC of primary mice was comparable to sBG-Imice (FIG. 13D). The amount of hβ-globin protein in the sBG650 mice,determined by HPLC analysis, was not significantly different from sBG-Imice (41±2.6% versus 43±3%, respectively), but was at least 2-foldhigher than that seen in the sBG mice (19±6%; P<0.01). Five months aftertransplant, secondary transplants were performed to generate CFU-S,which confirmed that the sBG650 vector restored insulator activitysimilar to that seen with sBG-I vector (FIG. 13E). The chromatinconfiguration over the core in sBG650 proviruses (FIG. 13F) showedrestoration of open chromatin patterns both over the insulator core andthe β-globin promoter, identical to those seen in the sBG-I proviruses(FIG. 10).

Example 26 Epigenetic Modifications in the 3′400 bp Region of cHS4 andits Interaction with the Core

The chromatin configuration of the distal 3′ 400 bp portion of cHS4 havenot been previously studied. The histone patterns were first analyzedover the 3′ 400 bp region (sBG3′400) when present alone (sBG3′400), orwhen in combination with the 5′ core (in sBG650 and sBG-I) (FIG. 14).The acetylation and methylation patterns of the histones in the 3′400region of sBG3′400 provirus (FIG. 14B) were similar to those seen in the250 bp core region in the sBGC provirus (FIG. 10). However, in sBG650and sBG-I proviruses, the 3′ 400 bp sequences had increased acetylationmarks and reduced repressive, showing once again, that the combinationof the proximal and distal ends of cHS4 is necessary for open chromatinpatterns. This effect was reminiscent of the ChIP analysis over the 5′core region or the β-globin promoter region in sBG-I (FIGS. 10D and F)or sBG650 (FIGS. 13F and G). Taken together, the genetic and epigeneticanalysis indicated that the 5′ and 3′ ends of the insulator werefunctioning as two cores, which interacted for epigenetic modificationsof chromatin on the insulator and promoter, to impart adequate insulatoractivity.

The 3′ 400 bp region, however, has no known CTCF or USF-1 motifs, thathave been shown to impart enhancer blocking and barrier activity,respectively, to cHS4. It is conceivable; however that CTCF and/or USF-1may perhaps be recruited to the 3′400 region. Using antibodies to USF-1and CTCF, chromatin was immunoprecipitated from sBGC, sBG3′400, sBG650and sBG-I proviruses from MEL clones. ChIP analysis was performed usingsemi-quantitative PCR and qPCR. When primers to the core region wereused to amplify ChIP products, CTCF and USF-1 recruitment to the 5′ coreregion was evident (FIG. 14C-D), as anticipated and shown previously.Interestingly, when 3′400 region primers were used to amplify the ChIPproducts, the sBG3′400 provirus showed enrichment for CTCF, albeit atsomewhat lower levels than that seen over the core region. More notably,however, the sBG650 and sBG-I proviruses showed enrichment both USF-1 atthe 3′ 400 bp region, an effect seen when both the proximal core and thedistal 400 bp sequences were present. The 3′ 400 bp region, when presentalone in sBG3′400, did not bind USF-1 (FIG. 14E-F). These data indicatethat the 3′ 400 bp region interacts with CTCF despite lack of the CCCTCconsensus, which may explain the “core-like” activity in this region andthe interaction between the 5′ core region and the 3′ 400 region of thecHS4 insulator (in sBG-I or sBG650) likely occurs via USF-1.

Example 27 Vector Titers with the 650 bp cHS4 Insulator

The 1.2 Kb cHS4 remarkably lowers titers of SIN-lentivirus vectors,limiting large-scale virus production for human trials. It has beenrecently shown that the mechanism of reduction in titers is specificallydue to the length of the insert in the 3′LTR. Compared to sBG, sBG650had very reasonable titers that were only 2.5±0.9 fold lower than sBG,in contrast to 10.4±2 fold lower titers of sBG-I (n=3). Therefore, thisoptimized insulator can be used for the design of safer gene therapyvectors which would provide uniform and therefore higher expression andbe scalable to large-scale production.

The full-length cHS4 insulator has been previously shown by us and byothers to protect viral vectors against chromosomal position effects.The profound deleterious effects on viral titers however, have precludedits utility. Attempts to use only the 5′ 250 bp of cHS4, characterizedto be the core of the insulator, have failed in viral vectors despitesignificant activity of the core in plasmid based systems, and loss ofinsulator activity with mutations in these regions.

Regions surrounding the cHS4 insulator and β-globin promoter have beenshown to constitutively higher marks of active chromatin in the nativelocation. The cHS4 prevents the spread of heterochromatin to theβ-globin domain, even when adjacent heterochromatin domains have highrepressive histone marks, H3K9me3 and H3K27me3. Clones carrying thesBG-I vector integrants showed an enrichment of the active chromatinmarks and a striking decrease in repressive chromatin marks over thecHS4 core compared to sBGC, sBG400 and sBG800 vectors, where nosignificant differences in these epigenetic marks were observed.

Mechanistically, the USF-1/2 element in the insulator has been shown torecruit histone modifying enzymes to the core, and interact with histonelysine methyl transferase SET7/9 and p300/CREB-bindingprotein-associated factor (PCAF), thus increasing active chromatinmarks. However, No such increase was observed in acH3, acH4 and H3K4me2over the core or the 3′ 400 bp when they flanked the transgene in thesBGC, sBG400, sBG800 and sBG3′400 vectors. This effect required thevector carrying the full length cHS4 (sBG-I, FIGS. 10 and 14) or boththe core and 3′400 bp combined sBG650 vector (FIGS. 13 and 14). ChIPanalysis over the hβ-globin promoter showed that compared to anuninsulated vector, the core alone reduced repressive chromatin marksover the promoter to some extent (FIG. 10F), which may account for thereduction in CV from vectors carrying the core. However, the core wasdependent on the 3′ 400 bp region and conversely, the 3′ 400 bp regiondependent on the core for the high degree of histone acetylation andabsent to minimal repressive marks over both these regions.

Models proposed to explain the effect of the cHS4 on surroundingchromatin include protection against transgene silencing by exclusion ofmethyl-CpG-binding proteins; indeed, cHS4 has been shown to blocksilencing by retroviral vectors. No extinction of β-globin expressionover time was observed, even with the uninsulated vector in mice, or MELclones maintained up to 6 months in culture (data not shown) This may bedue to several USF-1 elements in the β-globin LCR hypersensitive sites,that have been shown to interact with the E-box elements located in HS2and in the β-globin gene promoter. It is conceivable that thisresistance to silencing conferred by the LCR may override any activityseen with the cHS4 core. These results contrast those by Panell et althat retroviruses including those derived from HIV-1, dominantly silencea linked locus control region (LCR) beta-globin reporter gene intransgenic mice. Methylation was analyzed and it was subsequentlyreported that there was a lack of CpG methylation and extinction inexpression with erythroid-specific SIN-lentivirus vectors in vivo, inprimary and secondary recipients. This data suggests that in erythroidvectors, which otherwise resist silencing via promoter methylation, thefull-length cHS4 was able to modify the histone patterns over thetransgene promoter, and over itself to reduce position effects.

Intriguingly, the in silico analysis of the 3′ 400 bp region revealed noCTCF or USF1 binding sites, but sites for multiple known transcriptionfactors. Any of these transcription factors, or perhaps a novel proteinmay be the interacting partner with the CTCF and/or USF-1. CTCF directlyregulates the balance between active and repressive chromatin marks viabinding to the cohesin complex. This data reveals that the 3′ 400 bpregion can also interact with CTCF: although co-immunoprecipitate the3′400 bp and CTCF from the sBG3′400 provirus (FIG. 14C-F) wasunsuccessful.

Interestingly, the 3′400 bp co-immunoprecipated with USF-1 antibody onlywhen the 5′ core sequences were additionally present, suggesting thatUSF-1 likely forms a bridge between the 5′ and 3′ end of cHS4 to reduceposition effects. Whether elements within the 3′ 400 bp recruit histoneacetylases that bind USF-1 or cohesin and/or nucleophosphmin complexesto affect position effects would be important to determine.

Ultimately, a systematic genetic and epigenetic analysis of insulatoractivity of the cHS4 in vitro and in vivo was performed and novel“core-like” activity in the 3′ 400 bp was identified. The 3′ 400 bp ofcHS4, which contains no consensus sites for USF or CTCF, neverthelessbinds CTCF, while USF-1 appears to bind and bridge the 5′ core and the3′ 400 bp of cHS4. New vector systems flanked by the optimized ‘650 bp’cHS4 sequence, can provide excellent insulation of the transgene withoutsignificant loss in viral titers and have important safety and efficacyimplications for gene therapy.

Example 28 Materials and Methods-Lentivirus Vectors

All vectors were obtained by cloning the different insulator fragmentsinto NheI/EcoRV sites in the U3 3′LTR region of the lentivirus plasmid,as described. This plasmid carried the human (h)β-globin gene and itsregulatory elements (BG). All insulator fragments were amplified by PCRusing the insulator plasmid pJCI3-1 (kindly provided by Dr. GaryFelsenfeld, NIH, MD) and verified by sequencing, as described. Cloningof the hβ-globin vector with and without the 1.2 kb cHS4 insulator hasbeen described previously. The sBG1C vector was cloned by insertingEcoRI/XbaI 250 bp core insulator PCR product into sBG into BamHI/EcoRIrestriction sites of the pBS plasmid. A second copy of the 250 bp corewas then added into the pBS 1-core plasmid into EcoRI/KpnI sites, thusobtaining the pBS 2-core plasmid. The two tandem copies of the 250 bpcore were then isolated digesting the pBS-2core plasmid with KpnI/XbaI,and then cloned into the sBG vector, obtaining sBG2C. The sBG400 andsBG800 vectors were obtained by cloning the 2 PCR products into the sBGNheI/EcoRV sites. The vectors containing DNA spacers were obtainedamplifying different sizes of λ-phage DNA using the following primercombinations: spacerF1 and spacerR1, spacerF1 and spacerR2, amplifying150 bp, 550 bp X-DNA, respectively. ClaI/EcoRI digested PCR fragmentswere ligated into EcoRI/ClaI sites in the pBS-1 core plasmid, and 400 bpand 800 bp fragments from the pBS-1 core plasmid were restricted withHincII/XbaI and XbaI/XhoI, respectively, and cloned into NheI/EcoRVsites of sBG. Virus was produced by transient co-transfection of 293Tcells and titrated on MEL cells.

Example 29 Materials and Methods-Cell Lines

MEL cells and 293T cells were maintained in DMEM (Mediatech, Inc)supplemented with 10% heat-inactivated fetal bovine serum (FBS; U.S.Bio-technologies, Inc.) and differentiated as described. MEL cells weretransduced to achieve less than 5% transduction efficiency for each ofthe vectors tested and cloned. Approximately 400 clones, derived fromthree independent transductions from each vector were screened by PCRfor hβ-globin gene; positive clones were screened for an intactinsulator region. Clones thus identified were then subjected to qPCR forsingle integrants, expanded and cryopreserved. An entire set of cloneswas thawed, differentiated and analyzed concurrently by FACS.

Example 30 Materials and Methods-Murine Hematopoietic Stem CellTransduction and Transplants

Hbbth3/+ thalassemia mice were used for transplants. All animal studieswere done using protocols approved by the Institutional Animal Use andCare Committee. Enrichment of lineage-Sca-1+c-kit+ (LSK) hematopoieticstem/progenitor cells was performed on single cell suspension of bonemarrow by immunomagnetic separation and FACS sorting (details insupplementary Materials and Methods S1) LSK cells were transduced inStem Span (Stem Cell Technologies Inc, Vancouver, BC) with concentratedvector supernatants at an MOI of 10, twice at 12 h intervals aspreviously described. 10,000 transduced LSK cells were co-transplantedwith 2×105 LK cells into 10.75Gy irradiated thalassemia recipients.CFU-S assay: Discrete spleen colony forming units (CFU-S) were dissectedat day 12 after transplant of bone marrow cells from primary mice 24 wkafter transplant, as described earlier.

Example 31 Materials and Methods-Analysis for hβ-Globin Expression

Complete blood counts were performed on a Hemavet (Drew Scientific, Inc,Oxford, Conn., USA). Reticulocyte count was analyzed by staining 1 μl ofwhole blood with 200 μl of Retic-COUNT reagent (BD Biosciences, CA) andenumerated on the FACSCalibur (BD). Quantitative analysis of hβ-globinprotein in RBC was performed on hemolysates of blood by high performanceliquid chromatography (HPLC), as previously described and mRNA analysisquantified by real-time RT-PCR using validated primers and probesspecific to hβ-globin (ABI Biosystems) using murine a-globin fornormalization. FACS analysis following intracellular staining forhβ-globin was done as described before.

Example 32 Materials and Methods—Chromatin Immunoprecipitation (ChIP)

ChIP analysis was performed on MEL clones as described with minormodifications. Briefly, DNA samples from input and antibody-boundchromatin fraction were analyzed by qPCR using SYBR green (AppliedBiosystems) using primer sets in triplicate, and data analyzed aspreviously described. The enrichment ratio was determined by calculatingthe ratio of DNA-ChIP to DNA-input and histone modification datanormalized to the “no antibody” (IgG) control and primers correspondingto the necdin 5′ region and promoter region, as controls for repressedchromatin, to normalize the efficiency of immunoprecipitation. All theDNA-ChIP to DNA-input ratios were calculated as: 2[Ct (Input)−Ct (ChIP)]divided with [dilution rate (ChIP)/dilution rate (Input)]. Ct values ofall PCR products were determined by the SDS 1.2 software (AppliedBiosystems). Mean and SEM values were determined for the folddifference, and two-tailed paired t tests to determine statisticalsignificance (p<0.05).

Example 33 Materials and Methods-Integration Site Analysis

Ligation-mediated (LM) polymerase chain reaction was performed asdescribed by Modlich et al to map integration sites using primers andconditions described (Arumugam, Mol Ther 2009, in press citation).

Example 34 Materials and Methods-Statistical Analysis

Vectors were compared to the sBG vector Student's ‘t” test (unpaired andtwo tailed). ANOVA (Dunnett multiple comparison test) was also performedbetween groups for multiple comparisons. Data was expressed as mean±SEM.P<0.05 was considered significant.

Example 35 Self-Inactivating Lentiviruses Flanked by the 1.2 Kb ChickenHypersensitive Site-4 Insulator Element (cHS4) Provide Consistent,Improved Expression of Transgenes, but have Significantly Lower Titers

Self-inactivating lentiviruses flanked by the 1.2 Kb chickenhypersensitive site-4 insulator element (cHS4) provide consistent,improved expression of transgenes, but have significantly lower titers.Lengthening the lentivirus transgene cassette by an additional 1.2 Kb byan internal cassette caused no further reduction in titers. However,when cHS4 sequences or inert DNA spacers of increasing size were placedin the 3′LTR, infectious titers decreased proportional to the length ofthe insert. The stage of vector life-cycle affected by vectors carryingthe large cHS4 3′LTR insert was compared to a control vector: There wasno increase in read-through transcription with insertion of the 1.2 KbcHS4 in the 3′LTR. Equal amount of full-length viral mRNA was producedin packaging cells and viral assembly/packaging was unaffected,resulting in comparable amounts of intact virus particles produced byeither vectors. However, lentiviruses carrying cHS4 in the 3′LTR wereinefficiently processed following target-cell entry, with reducedreverse transcription and integration efficiency, and hence lowertransduction titers. Therefore, vectors with large insertions in the3′LTR are transcribed and packaged efficiently, but the LTR inserthinders viral-RNA processing and transduction of target cells. Thesestudies have important implications in design of integrating vectors.

Example 36 Increased Length of the Vector Genome by 1.2 Kb does notAffect Viral Titers

One objective of the study was to determine if reduction in titers bycHS4 was secondary to additional lengthening of the viral genomes in theotherwise large hp-LCR (BG) lentivirus vector. Large viral RNA genomesare known to be packaged less efficiently in integrating vectors.Replication competent gamma-retroviruses delete added sequences andrecombine to revert back to their original viral size. Ingamma-retrovirus vectors that exceed the natural size of the virus,reduction in titers occurs at multiple steps of the viral lifecycle—generation of full length genome, viral encapsidation/release andpost-entry recombination events. Notably, BG lentiviruses containtransgene inserts of ˜7 Kb, and therefore do not produce viral-RNAgenomes larger than the natural size/packaging capacity of the wild typeHIV-1 virus. In lentivirus vectors, however, lowering of viral titersfrom transgene inserts 6 Kb or larger has been shown to occur fromreduced packaging efficiency.

Uninsulated vectors BG and BGM were recently compared with analogousinsulated vectors BG-I and BGM-I for position effects. The BG lentivirusvector carries the hβ and LCR, while a similar vector BGM additionallycarries a PGK promoter driven methylguanine methyl transferase (P140K)cDNA (PGK-MGMT) insert downstream of the hp-LCR. The PGK-MGMT cassetteis 1.2 Kb in size. The BG-I and BGM-I vectors carry the 1.2 Kb cHS4insulator in the 3′LTR in addition. Virus was produced and processedidentically from all four vectors and infectious titers were determined,as previously described. The titers of the concentrated BG vector were2±0.5×10⁸ IU/mL, while that of BGM, carrying an additional 1.2 Kbinternal cassette were slightly higher at 5±0.8×10⁸ IU/mL (n=4). Incontrast, addition of the 1.2 Kb cHS4 in the 3′LTR to the BG vector,termed BG-I resulted in reduction in titers by nearly 6-fold to3.8±0.8×10⁷ IU/mL. A further addition of a 1.2 Kb PGK-MGMT internalcassette to the BG-I vector, termed BGM-I, did not reduce the titers anyfurther (FIG. 20B). These data indicate that cHS4 insertion into theLTR, and not overall viral genome size reduced viral titers. Ramezani etal observed a 3-fold reduction in lentivirus titers when the 1.2 Kb cHS4was inserted in lentivirus vectors encoding relatively small transgeneexpression cassettes (2 Kb in size or less). The present data isconsistent with their results, although indicating a 6-10 fold reductionin titers with the addition of cHS4. It was additionally observed in thepresent study that reduction in titers by insertion of insulatorelements in the LTR occurred by a distinct mechanism that was notdependent on the increased size of the viral genome.

Example 37 The Size of the Insert in the 3′LTR is Responsible forReduction in Titers

Although the LV vectors used did not exceed the natural size of theHIV-1 virus, the size of the cHS4 insert (1.2 Kb) exceeded the naturalsize of the wild type LTR (note that the wt LTR carries an additional400 bp U3 enhancer, which is deleted from the self-inactivating 3′LTR).Experimentation was conducted to determine whether lowering of viraltiters was due to lengthening of the SIN LTR beyond its natural capacity(400 bp), or whether titers were lower due to specific sequences in theinsulator, which may potentially affect viral-RNA folding/binding tocellular proteins and thus limit packaging. A series of p-globin vectorswere constructed in a self-inactivating lentivirus backbone, sSIN,carrying different length fragments of cHS4 in the 3′LTR (FIG. 15a ):the first 250 bp of the insulator, also called the core, a 400 bp cHS4fragment, matching the size of the U3 promoter/enhancer deletion in the3′ SIN LTR, and a 800 bp cHS4 fragment, to generate sBG^(C), sBG⁴⁰⁰,sBG⁸⁰⁰ vectors, respectively. These vectors were compared to ananalogous ‘uninsulated’ vector, sBG, and a vector carrying thefull-length 1.2 Kb insulator, sBG-I. In addition, a vector was clonedwith two copies of the core as tandem repeats (250 bp×2), sBG^(2C). ThecHS4 core has been shown to have 50% of enhancer blocking activity ofthe full length (1.2 Kb) insulator; the effect of the core has beenshown to be copy number-dependent, with tandem repeats of cHS4 corereported to have the same insulating capacity as the full length 1.2 KbcHS4.

Virus was generated from sBG, sBG^(C), sBG⁴⁰⁰, sBG^(2C), sBG⁸⁰⁰, sBG-Iplasmids by concurrent transient transfections and concentration, andtitered by flow cytometry of mouse erythroleukemia (MEL) cells infectedwith serial dilutions of the viruses, as described. MEL cells supportadult type globin production. Each experiment was replicated four times.

It was determined that as the size of the cHS4 insert in the 3′LTRincreased, viral titers dropped (FIG. 15b ). There was a slight, butstatistically significant reduction in titers with inserts of 250 bp and400 bp. However, titers fell sharply thereafter, proportional to thelength of the insulator fragment (FIG. 15b ). The titers of the vectorwith a 1.2 Kb full-length cHS4 insulator, sBG-I were an order ofmagnitude lower than the uninsulated control vector, sBG. Of note,sBG^(2C) vector, with a tandem repeat of two cHS4 core sequences (500 bpinsert) had titers similar to sBG⁸⁰⁰.

To ensure that reduction in titers was not from specific cHS4 sequencesbut an effect of the size of the LTR insert, three additional vectorswere constructed, sBG^(400-S), SBG^(800-S) and sBG^(1200-S). Thesevectors were analogous to sBG⁴⁰⁰, sBG⁸⁰⁰ and sBG-I, except that theycontained spacer elements from the 2 phage DNA downstream of the cHS4core to generate 3′ LTR inserts of 400 bp, 800 bp and 1.2 Kb,respectively (FIG. 15a ). The core cHS4 sequences were retained as thereduction in titers was minimal (and not observed in initialexperiments) with the core; and it was important to determine ifadditional sequences downstream of the core are necessary for optimalinsulator activity. The titers of the vectors containing DNA spacerswere identical to those containing similar sized cHS4 fragments, anddecreased with increasing size of the fragment in the 3′LTR (FIG. 15d ).These data show that lengthening of the 3′ LTR lowered titers and thiseffect was not from specific sequences in cHS4. It has been reportedthat HIV-1 RT is not a strongly processive polymerase; it dissociatesfrom its template frequently and the viral DNA is synthesized inrelatively short segments. Therefore, it is likely that as the size ofinsert in the U3 LTR increased, there was reduced processivity throughthe 3′ LTR.

Example 38 Recombination Occur with Repeat Elements in the 3′LTR

In order to detect if recombination events occurred in the LTRs frominsertion of 2 copies of the core or different size fragments in theLTR, ˜12-20 MEL cell clones transduced with the entire series ofinsulated vectors (sBG^(C), sBG⁴⁰⁰, sBG^(2C), sBG⁸⁰⁰ and sBG-I) weregenerated. All clones that had a single copy of integrated provirus wereidentified using qPCR, as previously described. The 250 bp core from thegenomic DNA of each clone was then amplified, by a standard PCR. Theinsulator core sequences could be amplified from clones derived from allvectors except those derived from sBG^(2C) transduced cells. In sBG^(2C)MEL clones, the insulator core was undetectable in 6 of 24 (25%) singlecopy clones by PCR, suggesting deletion of both tandem repeats of cHS4core sequences in the 5′ and 3′ LTR of the provirus (FIG. 20D). Tofurther analyze the frequency of recombined proviruses, a genomicSouthern blot analysis on sBG^(2C) transduced MEL cell pools wasperformed. Genomic DNA from sBG^(2C) and sBG-I MEL cell populations wasrestricted with an enzyme that cut within the LTRs. FIG. 15E shows theexpected lengths of the provirus with the sBG^(2C) vector and the sBG-Ivector, used as a control. While a single proviral band was seen insBG-I transduced MEL cells, the sBG^(2C) provirus in MEL cells showedloss of one or both copies of the cHS4 core sequences. Indeed, proviralbands containing two intact copies of the core were not detected at thelevel of sensitivity of Southern blot analysis. These data show thattandem repeats in sBG^(2C) recombined at a high frequency. The sBG^(2C)vector, therefore, had lower viral titers from recombination eventsduring reverse transcription, rather than the size of the LTR insert.These results were not unexpected, since repeat elements withingamma-retrovirus and lentivirus vectors have been shown to recombinefrequently.

Example 39 Steps in Vector Life-Cycle Affected by Large Inserts into the3′LTR

Large viral genomes in RNA vectors have been shown to be limited at thelevel of RNA packaging. In the present study, there was no effect ontiters with increasing the virus payload by 1.2 Kb, but titers decreasedwith increasing length of the insert in the LTR. Next, the mechanism bywhich this affected viral titers was explored. The following steps inthe viral life cycle were studied: 1) characteristics of viral-RNAproduced in packaging cells, 2) virus particle production, 3) post-entrysteps: reverse transcription, nuclear translocation, integration andproviral integrity. For all of these studies, the vector with thelargest insert, sBG-I was compared to the vector without the insulator,sBG.

Example 40 Insertion of cHS4 in the 3′LTR does not Alter the Quantity orQuality of Viral-RNA in Packaging Cells

Northern blot analysis was performed on RNA derived from the 293Tpackaging cells after transient transfection with sBG, sBG-I vectorplasmids, along with packaging plasmids (D8.9 and VSV-G). The blot wasprobed with hp fragment. FIG. 16 shows similar intensity viral-RNAtranscripts of the expected lengths of sBG and sBG-I vectors. The probenon-specifically probed the 28S and 18S RNA. Nevertheless, there were noadditional bands other than the full length-viral RNA of expectedlength, suggesting that no recombination or aberrant splicing occurredwith insertion of the insulator. Thus, viral-RNA was producedefficiently in packaging cells, independent of the presence of an insertin the LTR.

Example 41 Insertion of cHS4 in the 31TR does not Increase Read-ThroughTranscription

Experimentation was conducted to determine if the cHS4 insert upstreamof the viral polyadenylation signal in the LTR could impair transcripttermination of the viral RNA. Read-through transcripts have been shownto be excluded from encapsidation, and can lower viral titers. Althoughthe northern blot in FIG. 16 showed the expected size viral-RNA band andno extraneous transcripts, it has been shown that transcriptionalread-through is much less in lentivirus vectors, as compared togamma-retrovirus vectors, that may not be readily detectable via anorthern blot. Therefore a sensitive enzyme based assay was used tostudy read-through transcription.

Plasmid constructs were cloned, in which the wild type HIV-1 LTR, theSIN HIV-1 3′LTR with or without the insulator (from sBG-I or sBGvectors, respectively) were placed downstream of EF1-α promoter. Apromoter-less IRES-cre cassette was placed downstream of the LTRs, sothat cre expression would occur only from transcriptional read-throughfrom the LTR. An EF1a-IRES-cre plasmid served as a positive control.Equal amounts of these plasmids were transfected into the reporter cellline, TE26, which expresses β-galactosidase proportional to creexpression. A GFP plasmid was co-transfected with the read-throughplasmid constructs to normalize β-galactosidase activity fortransfection efficiency. A plasmid carrying the truncated rat nervegrowth factor receptor served as a negative control. A standard curvewas generated that showed a linear correlation of the amount of thepositive control IRES-cre plasmid transfected into cells and theβ-galactosidase activity measured by spectrophotometer. No significantincrease was observed in β-galactosidase activity from transfectedconstructs containing the insulated SIN lentivirus LTR, as compared tothose carrying the SIN LTR without the cHS4 insulator. The results fromthe β-galactosidase assay were identical when confirmed by Lac-Zstaining of TE26 cells plated on cover slips. These results showed thatthe insertion of cHS4 element upstream of the viral polyadenylationsignal did not increase read-through transcription from the LTR.

Example 42 Production of Viral Particles Containing Viral Genomes is notAffected by cHS4

To determine whether viral-RNA was encapsidated effectively intovirions, p24 levels, virus associated reverse transcriptase (RT)activity and viral-RNA levels (FIGS. 17a-c ) were measured. Virus wasgenerated in an identical manner concurrently with the two vectors, andconcentrated similarly in three separate experiments. To ensure purityof the viral preparation and lack of protein or plasmid contamination,virus was pelleted on a sucrose cushion and subjected to DNAse digestionfor these experiments. Lack of plasmid contamination was confirmed by aqPCR for the ampicillin resistance gene, present in the plasmidbackbone. The same volumes of virus preparation were then subjected top24 ELISA and virus-associated RT assays; and viral-RNA was extractedfor a dot-blot analysis. FIG. 17a shows that there was no difference inthe amount virus-associated RT between the two vectors. The p24 levelsin the sBG and sBG-I virus preparations were also similar (FIG. 17b ).In order to ensure sBG-I virions contained viral genomes, and were notempty viral like particles; virus was subjected to RNA dot-blotanalysis. FIGS. 17c-d shows one of two representative experiments. ViralRNA from sBG and sBG-I was loaded in duplicate in 4 different dilutionsof p24 (FIG. 17c ); and the intensity of the dots quantified byphosphoimager (FIG. 17d ). There were similar amount of viral mRNAencapsidated from either vector. These data suggest that insertion of a1.2 Kb fragment in the LTR did not affect packaging efficiency of viralmRNA or production of viral particles.

The present results with large inserts into the LTR are in contrast tothose by Sutton and colleagues where lentivirus vectors with lengthenedinternal transgene cassettes are inefficiently packaged into virions.Equal amounts of virus particles produced from the sBG and sBG-Ivectors, but significantly lower infectious/transduction titers suggestsa post-entry block of large LTR insert bearing viruses, resulting inless integrated units.

Example 43 Large LTR Inserts Affect Reverse Transcription andIntegration of Viral cDNA

Post-entry steps were investigated; including reverse transcription,nuclear translocation, integration and proviral integrity. ReverseTranscription: the steps of reverse transcription, location of qPCRprimers and probes and the viral DNA products are summarized in FIG. 18a. Reverse transcription initiates from the primer binding site near the5′ end of the genomic RNA, and minus strand synthesis proceeds to the 5′end of the genome (minus strand strong stop DNA (−sssDNA)). The newlyformed −sssDNA anneals to the 3′R region of the genome (first strandtransfer), minus-strand DNA synthesis resumes, accompanied by RNase Hdigestion of the viral RNA template. It has been shown that thesecondary structure of viral RNA at the 3′ end is a critical determinantfor the −sssDNA transfer, for the reverse transcription process to beefficient. Therefore, it is likely that presence of the insulator/aninsert in the U3 region of the 3′ LTR would alter the secondarystructure of the region involved in this complex process, resulting inoverall decreased reverse transcription efficiency.

To assess reverse transcription efficiency, MEL cells were infected withequal amounts of sBG and sBG-I viral particles, based upon p24 levels,and cells collected at different time points post infection. Absence ofplasmid contamination was confirmed by a qPCR for the ampicillinresistance gene present in the plasmid backbone (data not shown).Kinetics of early reverse transcription (production of −sssDNA) werestudied using primers and probe spanning the R/U5 region (FIG. 18b ). Asexpected, there was no difference detected in the kinetics between thetwo viruses, since the 5′ ends of sBG or sBG-I viral RNA were identical.Nevertheless, the data validated that qPCR accurately determined viralreverse transcription.

It is conceivable, however, that when RT switches templates (minusstrand jump) to reverse transcribe the 3′ LTR, alteration of secondarystructure from the presence of an insert in the U3 region would reducereverse transcription products. Quantitative PCRs amplifying the U3/Rand ψ regions were performed to quantify the amount of intermediate andlate reverse transcribed viral cDNA in cells infected with sBG and sBG-Ivectors, respectively (FIGS. 18c-d ). It was discovered that RTefficiency soon after the first strand transfer was impaired. Notably,the U3/R primers amplified viral DNA that was reverse transcribed beforethe insulator sequences, suggesting that insert in the 3′LTR affectedreverse transcription by altering or “poisoning” the 3′LTR. Indeed, theinefficiency in intermediate RT product formation was similar to thatseen with late RT products. In both analysis, the peak of viral cDNAsynthesis occurred at 12 h for the uninsulated vector sBG and thengradually decreased, consistent with integration of viral cDNA, andpreviously reported kinetics of reverse transcription. The amount ofviral DNA from the insulated vector sBG-I was lower post-entry comparedto sBG by about 2-fold at all time points, as early as 6 hourspost-target cell entry. These data strongly suggest that reversetranscription after the minus strand jump was rate-limiting in the sBG-Ivector.

Nuclear translocation: After the viral DNA is synthesized in thecytoplasm, it is translocated into the nucleus of infected cells, whereit can be found as linear DNA or circular DNA (1-LTR and 2-LTR circles)(FIG. 18a ). The linear form is circularized at the LTRs and is thedirect precursor of the integration process; 1-LTR and 2-LTR circles,instead, are abortive products of homologous recombination andnon-homologous DNA end joining, respectively. However, 1LTR and 2LTRcircles are specifically localized in the nucleus, and are used as amarker for nuclear translocation. Presence of an insert in the LTR oflentiviruses can possibly interfere with the pre-integration complex(PIC) formation and the nuclear translocation of the viral DNA can lowertransduction titers. It has been shown indeed that PIC complexes bindHIV LTR in the cytoplasm, and they are responsible for the transport tothe nucleus and the integration of the cDNA into the genome of infectedcells.

In order to detect the nuclear translocation, the amount of 2-LTRcircles in both vectors were analyzed using a qPCR on DNA from infectedMEL cells at different time points in sBG versus sBG-I infected cells.As shown in FIG. 19a , the amounts of 2-LTR circles were notsignificantly different between the two vectors at early time points.However, at 48 h after infection, the peak at which 2-LTR circles arenormally detected, 2-LTR circles were 6.7 times higher in sBG infectedcells, but were barely at the detection limit in sBG-I infected cells.Later time points (72 and 96 hours) were also analyzed, but no delay wasdetermined in the kinetics of 2LTR circle formation in the insulatedvectors. Indeed, the 2-LTR circles were barely detectable by qPCR in thesBG-I infected cells after 24 hours. These data suggested that nucleartranslocation was likely reduced due to presence of the large U3 insert.

Integration: It is also conceivable, however, that two copies of largeU3 inserts provide a template for homologous recombination, and the rateof homologous recombination between the two LTRs prior to integrationincreases, resulting in more 1-LTR circles and reduced 2-LTR circles (asproposed in the cartoon in FIG. 20). This would decrease the amount oftemplate available for integration. Due to the nature of reversetranscribed viral cDNA with an insulated and uninsulated vector, 1LTRcircles cannot be quantified by a PCR-based technique. Therefore, aSouthern blot analysis was performed to detect linear viral cDNA, 1-LTRand 2-LTR circles at 72 hours post infection with equal amounts of sBGand sBG-I (quantified using p24 levels) (FIG. 19b ). The Southern blotanalysis showed that (i) the linear form of reverse transcribed viralcDNA, the form that integrates, was undetectable in the sBG-I lane atthe sensitivity of Southern blot analysis, while it was readilydetectable in the sBG lane. (ii) The 2-LTR circles were alsoundetectable in the Southern analysis in the sBG-I lane, but detectablein the sBG lane, corroborating the qPCR data on 2-LTR circles. (iii)However, large amount of 1-LTR circles were present in sBG-I lane,similar in amount to those seen in the sBG lane. The relative ratios oflinear, 1- and 2-LTR circles in sBG versus sBG-I lanes suggested thatthere was increased homologous recombination of the sBG-I viral DNA.Indeed, these data indicated that nuclear translocation was not affectedto any major extent by the U3 insert. But after the reverse transcribedcDNA entered the nucleus, increased 1-LTR circles, representing abortiverecombinant integration products were formed due to the large LTR insertand therefore, integration was reduced.

It is conceivable that the integration machinery is also directlyaffected by the presence of foreign sequences in the LTR. Therefore, sBGand sBG-I viruses were packaged using an integrase defective packagingplasmid, so that effect of the insulator on reverse transcription,nuclear localization, and 1LTR circle formation could be studiedindependent of integration. The same analysis was performed as withactive integrase containing viruses: a q-PCR to study the late reversetranscription product (using psi primers), 2LTR circles and a genomicSouthern blot analysis to determine 1LTR circles and other forms ofviral cDNA. The results were identical to those seen with sBG and sBG-Ipackaged with active integrase (shown in FIG. 19b ): the same reductionwas observed in late RT products and 2LTR circles by qPCR, but increased1LTR circles by genomic Southern analysis (data not shown). Therefore,sequences inserted into the lentivirus LTR interfered mainly with thereverse transcription process, and increased the frequency of homologousrecombination by a mechanism independent of the integrase machinery.

Finally, the integrated sBG and sBG-I provirus were analyzed forstability of transmission and efficiency of integration. The Southernblot analysis in FIG. 19b shows the integrated DNA as a smear, that isof higher intensity in the sBG than the sBG-I lane. In order to confirmand quantify integration, MEL cells were transduced with same amount ofp24 levels of sBG or sBG-I virus, cultured for 21 days and a qPCR andSouthern blot analysis were performed to compare proviral integrationefficiency and stability (FIG. 19c ). There were 6.2 proviral copies percell in sBG MEL cell population by qPCR, while only 0.8 proviral copieswere detected in sBG-I MEL cells, a 7.8-fold difference which isconsistent with differences seen in transduction titers between the twovectors. Next, DNA was restricted with Afl-II, an enzyme that cutswithin the LTRs (FIG. 19c , left panel). Consistent with transductiontiters and qPCR, the amount of integrated sBG-I provirus was 8-fold lessthan sBG, as indicated by phosphoimager quantification of the Southernblot bands (FIG. 19c ). The sBG-I vector did not recombine, as shown bythe single proviral band of the expected size. Next, the full lengthinsulator was detected by PCR in all single copy clones of sBG-Itransduced MEL cells (FIG. 20D). Therefore, the linear sBG-I cDNA,albeit inefficiently formed, integrated as an intact provirus.

The overall reduced viral integration was primarily from a combinationof inefficient reverse transcription and increased homologousrecombination that hinder the availability of proviral DNA forintegration. Since insulators are important for generating viral vectorsthat would be safe and provide consistent predictable expression, it isimportant to find a solution to the problem of low viral titers withinsulated viruses. One way to overcome the problem would be to flank theinternal expression cassette with cHS4 on either end, since furtherlengthening of the internal cassette did not decrease titers. However,this approach was not tried because repeat elements within retrovirusesare known to result in recombination. Since HIV RT is known to have lowprocessivity and frequently dissociate from its template, an attempt wasmade to increase the amount of RT delivered per vector particle, toassess if that would improve reverse transcription from large LTRinserts. RT was co-packaged in the virions as vpr-RT fusion protein. Nosignificant increase in titers was observed when providing more RT inthe virion. The next step was an attempt to increase the integrase (IN)per virion using the same strategy, and copackaged RT-IN-vpr fusionprotein in the virion. There was a slight increase in titers providingRT-IN in the viral particle, but the difference was not significant.

Next, a detailed structure-function analysis of the 1.2 Kb cHS4insulator was performed and a defined 650 bp sequences were determinedas the minimum necessary sequences for full insulation effect. Thetiters of sBG⁶⁵⁰ were 3.6×10⁸IU/mL, compared to a titer of 8.2×10⁸ IU/mLand 9.8×10⁷ IU/mL of the sBG and sBG-I vectors (FIG. 20C). Vectors withthe 650 bp insert had very reasonable viral titers (2.2-fold lowertiters than the uninsulated vector sBG, as compared to 9-10-fold lowertiters of sBG-I) with no loss of insulator activity.

Ultimately it was determined that low transduction titers were not froman increase in size of the provirus, but increased length of the 3′LTR.The quantity and quality of viral RNA genomes produced were unaffectedand viral-RNA encapsidation/packaging was comparable in vectors with andwithout a 1.2 Kb LTR insert. Reduced viral titers occurred frompost-entry steps, from inefficient reverse transcription, increasedhomologous recombination in the LTRs of viral DNA, making less viral DNAavailable for integration. Improvements in vector design were made byincluding smaller insulator inserts that contained essential elementsnecessary for optimal insulator activity.

The present studies have important implications for future design ofvectors with inserts within the 3′LTR, given the usefulness of chromatininsulator elements, customized lineage specific LTR vectors or doublecopy vectors.

Example 44 Vector Constructs

The cloning of the BG, BGM, BG-I and BGM-I vectors has been previouslydescribed. All other vectors were cloned into the sSIN backbone (detailsprovided in Urbinati F, Xia P and Malik P, manuscript in review). Allthe vectors were obtained cloning the different insulator fragments intoa unique Nhe I/EcoR V site was inserted in the U3 3′LTR region of thesSIN LV vector plasmid, which carried the human beta-globin gene and thehypersensitive site 2, 3 and 4 fragments, as previously described.Insulator fragments were amplified by PCR using the insulator plasmidpJCI3-1 as a template. All amplicons were sequenced following the PCR,and after insertion into the 3′LTR. The cloning of the uninsulatedbeta-globin vector and one that carrying the full length 1.2 Kb cHS4insulator has been described previously. Briefly, the 1.2 Kb insulatorfragment was obtained by digesting pJCI3-1 plasmid with Xba I and clonedinto the Nhe I/EcoR V restriction site of sBG. sBG^(C) was clonedinserting into sBG vector the fragment EcoR I/Xba I containing the 250bp core from the pBS 1 core plasmid. The latter was obtained cloning the250 bp core Insulator PCR product (using Core 1F and Core 1R primers, asdescribed herein) into BamH I/EcoR I restriction sites of a pBS plasmid.A second copy of the 250 bp core was then added into the pBS 1 coreplasmid, cloning into EcoR I/Kpn I sites the PCR product (Core 2F andCore 2R), obtaining the pBS 2 core plasmid. 2 tandem copies of the 250bp core were then isolated digesting the latter plasmid with Kpn I/XbaI, and then cloned into the sBG vector, obtaining sBG^(2C). The sBG⁴⁰⁰and sBG⁸⁰⁰ vectors were obtained cloning the 2 PCR products (using InsFand Ins400R primers and InsF and Ins800R primers, respectively) into thesBG Nhe I/EcoR V sites. sBG650 vector was obtained cloning the 3′ 400fragment of the insulator in EcoRV/BspEI sites of sBG^(1c) vector. The3′ 400 fragment was PCR amplified from the plasmid pJCI3-1 using thefollowing primers: 3′ 400 R (BspEI) and 3′ 400 F (EcoRV).

The vectors containing the λ DNA spacers were obtained amplifyingdifferent size λ phage DNA using the following primer combinations:spacerF1 and spacerR1, spacerF1 and spacerR2 and spacerF1 and spacerR3amplifying a 150 bp, 550 bp and 950 bp λ DNA fragments, respectively.The three PCR fragments were digested with Cla I and EcoR I restrictionenzymes and ligated into EcoR I/Cla I sites in the pBS-1 core plasmid,The 400 bp, 800 bp and 1200 bp fragments were digested from the pBS-1core plasmid with HincII and XbaI for the 400 bp fragment, and with XbaI and Xho I for the remaining two fragments, and cloned into the EcoRV/Nhe I restriction sites in the sBG vector. All the vectors cloned wereconfirmed by sequencing. The list of all the primers is available in(FIG. 20E).

Example 45 Cell Lines

Murine erythroleukemia cell (MEL) line and 293T cells were maintained inDulbecco modified Eagle Medium (DMEM, Mediatech, Inc) supplemented with10% heat inactivated fetal bovine serum (FBS) (U.S. Bio-technologies,Inc.). MEL cells were induced to differentiate in DMEM containing 20%FBS and 5 mM N, N′-hexamethylene bisacetamide (Sigma), as previouslydescribed. To derive single integrant clones, transduced MEL cells werecloned and clones were screened for β-globin sequences by PCR toidentify transduced clones. Single copy clones were identified by qPCRfor lentivirus y-sequences, and a PCR for the cHS4 core sequences wasperformed on the single integrant clones to confirm presence ofinsulator sequences in the provirus.

Example 46 HbA Staining and FACS Analysis

The staining using the anti-human HbA antibody was as previouslydescribed. Briefly, cells were fixed in 4% paraformaldehyde for 60minutes at room temperature, washed once with phosphate-buffered saline(PBS), and the pellet resuspended in 100% methanol for 5 minutes. Thefixed cells were then washed with PBS, and nonspecific antibody (Ab)binding was blocked using 5% nonfat dry milk for 10 minutes at roomtemperature. Subsequently, cells were washed in PBS, pelleted, andpermeabilized. The cells were divided into 2 tubes and stained witheither anti-Zeta globin-fluorescein isothiocyanate (FITC) (1 μg/10⁶cells) as a negative control or anti-HbA-FITC (0.1 μg/10⁶ cells) (PerkinElmer) for 30 minutes at room temperature in the dark. Unbound Ab wasremoved by a final wash with PBS before they were analyzed on FACSCalibur (Becton Dickinson).

Example 47 Virus Production

Virus was produced by transient cotransfection of 293T cells, aspreviously described, using the vector plasmids, the packaging (Δ8.9 orΔ8.2 for active or inactive integrase respectively) and the VSV-Genvelope plasmids; virus-containing supernatant was collected at 60hours after transfection and concentrated by ultracentrifugation. Allvectors in an experiment were packaged simultaneously. Virus was treatedwith DNase and/or DpnI to remove plasmid DNA contamination and layeredon a 20% sucrose cushion to obtain purified viral particles for specificexperiments on vector life cycle indicated in the results. Virus wasconcentrated 1400-fold from all viral supernatants afterultracentrifugation at 25,000 rpm for 90 minutes. Viral titers weredetermined by infecting mouse erythroleukemia (MEL) cells with serialdilutions of concentrated virus, differentiating them, and analyzingthem for HbA expression by fluorescence-activated cell-sorter scanner(FACS).

Example 48 Northern Blot

Total RNA was extracted from 293T cells using RNA-STAT (Tel-Test, INC,Texas), 72 hours after transfection. Northern Blot was then performedaccording to standard protocol. The blot was hybridized with a ³²-Plabeled β-globin probe.

Example 49 RNA Dot Blot

Viral-RNA was extracted from same volumes of concentrated viruses usingthe QIAamp Viral RNA Mini Kit (Qiagen, Valencia, Calif.) following themanufacturer's instructions. Briefly the virus was lysed under a highlydenaturing condition and then bound to a silica-gel-based membrane. Twowashing steps efficiently washed away contaminants and v-RNA was elutedin 30 μl of DEPC-H2O. After elution viral-RNA was treated for 20 min. atroom temperature with amplification grade DNAse I (Invitrogen). DNasewas inactivated incubating the sample at 65°. Viral RNA was thendenatured in 3 volumes of denaturation buffer (65% formamide, 8%formaldehyde, MOPS 1×) for 15 min at 65°. After denaturation 2 volumesof ice-cold 20×SSC were added and the RNA was bound to a nylon membraneby aspiration through a dot-blot apparatus. The blot was hybridized witha ³²-P labeled β-globin specific probe and a film was exposed overnight.Quantification of the dots was performed with a phosphoimager (Biorad,Hercules, Calif.).

Example 50 Reverse Transcriptase Assay

Concentrated virus (1 μL), and serial dilutions (1:10, 1:100, 1:1000)were lysed and processed following the “Reverse transcriptase (RT)assay, colorimetric” Kit (Roche) protocol. Briefly concentrated viralparticles were lysed with lysis buffer and viral-RNA reverse transcribedusing digoxigenin and biotin-labeled nucleotides. The detection andquantification of synthesized DNA as a parameter of RT activity followeda sandwich ELISA protocol: biotin-labeled DNA was bound to the surfaceof microplate modules that were pre-coated with streptavidin. In thenext step, an antibody to digoxigenin, conjugated to peroxidase(anti-DIG-POD), was bound to the digoxigenin-labeled DNA. In the finalstep, the peroxidase substrate ABTS was added, that resulted in acolored reaction product that was quantified using an ELISA reader at awavelength of 405 nm. The amount of colored product directly correlatedto the level of RT activity in the sample.

Example 51 P24 Assay

P24 antigen concentration was determined by HIV-1 p24 Antigen EIA Kit(Beckman Coulter). Briefly, serially diluted virus was lysed andincubated onto p24 antigen coated microwells, and washed followingmanufacturer's protocol. Color absorbance was measured using aspectrophotometer at a wavelength of 450 nm. p24 assay was performed induplicate.

Example 52 Southern Blot

To analyze the integrity of the provirus we infected MEL cells, expandedthem for 21 days and extracted DNA using Qiagen Blood and Cell cultureDNA Mini Kit (Qiagen). 10 μg of DNA was digested with Afl II, an enzymethat cuts in the LTRs. To determine presence of viral linear DNA,genomic DNA was extracted 72 h after infection of MEL cells andrestricted with Stu I, an enzyme that cuts twice within the provirus.The DNA was separated on a 0.8% agarose gel, transfer to a nylonmembrane, and probed overnight with a β-globin fragment.

Example 53 Real Time PCR for RT Products and 2LTR Circle

The same amount of p24 was used to transduce MEL cells with sBG andsBG-I vectors, in DMEM media, in the presence of 8 μg/mL polybrene.Cells were harvested at different time point (0.5 h, 3 h, 6 h, 8 h, 12h, 24 h, 48 h, 72 h) and DNA extracted using Qiagen Blood and Cellculture DNA Mini Kit (Qiagen). Genomic DNA (50 ng) from a single copyMEL clone (confirmed by Southern for a single integrant) was dilutedwith untransduced DNA to generate copy number standards (1-0.016copies/cell). The primers and the probe for RT product were designedusing the Primer Express Software from Applied Biosystems, Foster City,Calif. Primers and probe sequence for early RT products (R/U5) qPCRassay are: forward primer 5′-GAACCCACTGCTTAAGCCTCAA-3′, reverse primer:5′-ACAGACGGGCACACACTACTTG-3′ The reaction was carried out with TaqManMGB Probe: 5′-AAAGCTTGCCTTGAGTGC-3′. Primers and probe sequence forintermediate RT products (U3/R) qPCR assay are: forward primer5′-CCCAGGCTCAGATCTGGTCTAA-3′, reverse primer:5′-TGTGAAATTTGTGATGCTATTGCTT-3′ The reaction was carried out with TaqManMGB Probe: 5′-AGACCCAGTACAAGCAAAAAGCAGACCGG-3′. For the late RT productassay (psi) the primers were designed to recognize the ψ region of theprovirus: forward primer: 5′-ACCTGAAAGCGAAAGGCAAAC-3′, reverse primer:5′-AGAAGGAGAGAGATGGGTGCG-3′. The reaction was carried out with TaqManProbe: 5′-AGCTCTCTCGACGCAGGACTCGGC-3′ with TAMRA dye as quencher.Normalization for loading was carried out using mouse apoB genecontrols. The cycling conditions were 2 min at 50° C. and 10 min at 95°C., then 40 cycles of 95° C. for 15s and 60° C. for 1 min. The primersand probe for 2LTR circle were as previously described. The PCR mixturewas thermo cycled according to the thermal cycler protocol for 96 wellplates in Applied Biosystems 7900HT Fast Real-Time PCR System Base Unit.

Example 54 Generally

Sickle cell anemia (SCA) results from a point mutation in the-globingene (β^(S)), resulting in sickle hemoglobin (HbS). HbS polymerizes upondeoxygenation resulting in sickle-shaped RBCs that occludemicrovasculature. Patients with SCA have intermittent acute vascularocclusions and cumulative organ damage, reducing the life span to 42 to58.5 years. Besides sickling, excessive hemolysis and a state of chronicinflammation exist. SCA patients account for approximately 75,000hospitalizations per year, resulting in an estimated annual expenditureof $1.2 billion dollars in the United States alone. Worldwide, SCA issecond only to thalassemia in incidence of monogenic disorders, withmore than 200,000 children born annually in Africa.

Current therapies include supportive care for episodic sickling, chronictransfusions with iron chelation, and hydroxyurea to induce fetalhemoglobin (HbF). These therapies impact disease morbidity, but theireffectiveness is variable and dependent on compliance to an indefinitetreatment regimen. A matched allogeneic hematopoietic stem cell (HSC)transplantation is curative, but restricted by the availability ofmatched related donors 5 and has potential serious complications. Ameta-analysis of 187 SCA transplantations shows 6% to 7%conditioning-related peritransplantation mortality, 7% to 10% acuterejection, and 13% to 20% chronic graft-versus host disease (GVHD) inrecipients.

Gene therapy of autologous HSCs followed by transplantation could resultin a one-time cure, avoid adverse immunologic consequences, and not belimited by availability of donors; it may also not requiremyeloablative-conditioning regimens, and thereby have lower toxicity.The amount of HbF/anti-sickling globin required to correct SCA via atransgene is unknown.

Expression of HbF postnatally can be therapeutic, as is evident by theprotective effect of HbF in neonatal sickle RBCs and in patients withhereditary persistence of HbF and SCA. The proportion of geneticallycorrected HSCs, the amount of exogenously expressed HbF, and theproportion of F cells that will correct the pathophysiology are unknown.Complete correction of human thalassemia major in vitro, and inxenografted mice in vivo, with a lentivirus vector carrying the β-globingene and locus control region (LCR) elements has been demonstrated. Inthis report, this β-globin lentivirus vector was modified to encodeγ-globin exons and murine sickle HSCs were transduced. Functionalcorrection was characterized first, with a careful and detailedquantification of RBC sickling, half-life, and deformability, withsickle to normal transplantations and high HbF production to defineparameters of correction. Next, using reduced-intensity conditioning andvarying the percentage of transduced HSCs, transplantations wereperformed on sickle mice with significant organ damage and demonstratethe proportions of (1) genetically corrected HSCs, (2) HbF, and (3) Fcells, and (4) percentage of HbF/F cell required for correction of thesickle RBC and amelioration of organ damage in SCA.

Example 55 Vector

It has been demonstrated that a β-γ-globin hybrid gene carryinglentivirus vector, I8H β/γW,11 expresses high γ-globin mRNA in erythroidcells expressing “adultlike” globins. All β-globin coding sequences werechanged to γ-globin using site-directed mutagenesis and the γ-β-globinhybrid gene, and LCR elements were cloned in reverse orientation to theviral transcriptional unit to generate sGbG lentivirus vector. Virus wasmade with cotransfection of 293T cells.

Example 56 Murine HSC Enrichment

Bone marrow from 6- to 20-week-old BERK sickle mice was harvested andlineage depleted with biotinylated CD5, CD8, B220, Mac-1, CD11b,Gr-1,and TER-119 antibodies and magnetic beads. The bead-free cells werestained with antibodies to Sca-1, c-kit. Cells that were 7-AAD⁻,Lineage⁻, c-kit⁺ then Sca-1⁺ (LSK cells) were sorted on FACSVantage(BDBiosciences). All experiments using Berkeley transgenic sickle miceand C57/BL6 mice were performed according to protocols approved by theCincinnati Children's Hospital Medical Center.

Example 57 Gene Transfer and Bone Marrow Transplantation

Myeloablative transplantations were performed from BERK3C57B1/6 micebecause of ease of transplantation and ready availability of normalrecipients (9.5^(+/−) 0.6 weeks old) after 11.75 Gy radiation. Radiationcontrol experiments showed that BERK mice receiving 8 to 9 Gy radiationsurvived without receiving LSK cells; and the lethal dose was lower thanin C57Bl/6 mice. BERK mice receiving more than 10.5 Gy died when no LSKcells were given; those given LSK rescue survived long term. BERK miceare difficult to breed in large numbers at a given time, therefore 2mice/radiation dose level were to determine the sublethal dose. All BERKrecipients (12.9^(+/−) 0.4 weeks old) received 3 peritransplantation RBCtransfusions (days 1-7). Organ pathology in BERK recipients 1 year aftertransplantation was compared with 12-week-old BERK mice that did notundergo transplantation. The radiation was higher than classical reducedintensity radiation dose of 4 Gy to allow a large degree of donor HSCchimerism. A range of MOI was used to vary the proportion of transduceddonor HSCs in the graft. LSK cells were prestimulated overnight andtransduced twice at an MOI of 30 for BERK3C57BL/6 transplants and MOI of30 to 100 for BERK→BERK transplants for 22 to 24 hours; 10,000 to 24,000LSK cells and untransduced LK cells were cotransplanted into recipientC57BL/6 or BERK mice.

Example 58 Copy Number Analysis

Copy number analysis was done on genomic DNA by real-time polymerasechain reaction using primers and probes described previously.

Example 59 Hematologic Analysis

Hematologic analysis was obtained on Hemavet 950FS (Drew Scientific)under mouse settings. Reticulocyte analysis was performed as follows:0.1 μL blood and 200 μL BD Retic-COUNT Reagent were mixed (BectonDickinson), incubated at room temperature for 30 minutes, and analyzedby fluorescence-activated cell sorting (FACS).

Example 60 Hemoglobin Analysis

Hemoglobin electrophoresis was performed on cellulose acetate plates, asdescribed previously. Ion exchange high-performance liquidchromatography (HPLC) was performed with an Alliance 2690 HPLC machine(Waters) using a PolyCATAcolumn (item no. 3.54CT0510; Poly LC Inc).

Example 61 Red Blood Cell Functional Analysis

Irreversibly sickled cells (ISCs) were enumerated by scoring 500 RBCs inconsecutive fields. Graded deoxygenation was performed using tonometry.RBC deformability was determined using a laser-assisted opticalrotational cell analyzer (LORCA; RR Mechatronics).

Example 62 RBC Half-Life

Mice were injected with 3 mg Sulfo-NHS biotin (Sigma) in 300 μL PBS as 2separate injections 1 hour apart; 2 to 5 μL blood was drawn at serialtimes, and stained with APC-Cy7-conjugated streptavidin.

Example 63 Histology

Spleen, liver, bones, brain, and kidney were harvested and placed in 5mL of 10% formalin. Paraffin blocks were sectioned and stained withhematoxylin and eosin.

Example 64 High HbF after Gene Therapy and Myeloablative TransplantationCorrects SCA

The sG^(b)G vector carries γ-globin exons and β-globin noncoding andregulatory regions. Based upon a previously studied sBG vector, whichexpresses high levels of human β-globin, 13 sG^(b)G-transduced LSK cellsfrom Berkeley sickle (BERK) mice were transplanted into lethallyirradiated (myeloablated) normal C57Bl/6J mice (termed sG^(b)G mice).Mock transductions on BERK LSK cells from the same bone marrow poolfollowed by transplantation resulted in mice with SCA. The majority ofRBCs in sG^(b)G mice expressed HbF. Only sG^(b)G mice with 100% donor(HbS⁺) RBCs, with no evidence of residual recipient murine hemoglobin byelectrophoresis and HPLC, were analyzed for hematologic, functional, andpathologic analysis. sG^(b)G mice with a small proportion of recipientmurine RBCs, were used only to assess HbF/vector copy and frequency oftransduced HSCs. The percentage of HbF (HbF/HbS+HbF) in blood,quantified by FACS, was approximately 40% in primary mice followed for 6months and in secondary recipients followed for 7.5 months (FIG. 21A).Two-thirds of RBCs were F cells; their proportion was also stable inprimary and secondary recipients (FIG. 21B). The proportion of F cellsand vector copies correlated with HbF (FIG. 21C-D). Taken together,these data show significant HbF expression from the sG^(b)G vector inthe majority of RBC with stable long-term expression.

Example 65 High Levels of HbF Result in Sustained Hematologic Correction

FIG. 21E shows improvement of hematologic parameters in sG^(b)G mice.The proportion of reticulocytes decreased from approximately 50% in mockmice to approximately 15% in sG^(b)G mice (P<0.005; FIG. 22A). There wascorrection of anemia by 12 weeks, which persisted throughout theposttransplantation period (FIG. 22B-C)}.

High white blood cell (WBC) counts in humans with SCA and BERK micereflect the baseline inflammation in this disease. WBC returned tonormal levels in sG^(b)G mice (FIG. 22D; FIG. 21E).

Notably, WBC counts were lower in the mock mice compared with BERK micethat did not undergo transplantation, likely because in the former,sickle HSCs were transplanted into a normal “noninflamed” C57/BL6background. Indeed, 6 weeks after transplantation, WBC counts in mockgroup of mice were nearly normal, then gradually rose to high levelsseen in SCA (FIG. 22D) Overall, hematologic parameters showed markedimprovement to near normal levels, and improvement was stable over aprolonged period in primary and secondary sGbG mice. The degree ofcorrection correlated with the proportion of F cells (FIG. 22E-H) andHbF (data not shown). High levels of HbF improve the functionalparameters of RBCs in sickle mice. (1) Sickling: The irreversiblysickled cells (ISCs) were significantly reduced to 2.3% plus or minus0.7% in sG^(b)G mice, compared with 12% plus or minus 0.8% in BERKcontrols and 10.2% plus or minus 0.3% in mock mice (FIG. 23A-B).Deoxygenation of blood from a representative sG^(b)G mouse shows adramatic reduction in sickling (FIG. 23C). A systematic quantificationshowed a marked decrease in the proportion of sickle RBCs in sG^(b)Gmice with increasing hypoxia (FIG. 23D). (2) RBC membrane deformability:Normal RBCs deform readily at low shear stress (3 Pascals [Pa]),representative of shear stress in small vessels. Sickle RBCs haverelatively rigid membranes with remarkably reduced deformability even athigh shear stress (28 Pa; representative of shear stress in largevessels). There was markedly improved deformability of RBCs of sG^(b)Gmice, although it did not achieve normal levels (FIG. 23E). This mayreflect the proportion of circulating sickle RBCs that did not containHbF. (3) RBC survival: Survival of human sickle RBCs is an order ofmagnitude less than normal RBCs. The time to 50% reduction (half-life)in sG^(b)G and mock/BERK sickle mice was measured. The overall survivalof the sG^(b)G RBCs was markedly improved, with the time to 50%reduction approximately 4 times longer in RBCs from sG^(b)G micecompared with BERK or mock mice (FIG. 23F). (4) RBC hemolysis: RBChemolysis detected by measuring lactate dehydrogenase (LDH) in blood wasreduced from 2706 plus or minus 148 mg/dL in mock mice to 1286 plus orminus 345 mg/mL in sG^(b)G mice (n=5; P<0.004).

Example 66 High Levels of HbF Prevent Chronic Organ Damage Associatedwith SCA

Bone marrow, spleen, liver, and kidneys at 24 weeks showed completeprevention of organ pathology. There was reduced erythroid hyperplasiain bone marrow and spleen, decreased spleen size, and preservation ofthe splenic follicular architecture, compared with obliteratedfollicular architecture from the severe erythroid hyperplasia in mockmice. The focal tubular atrophy and segmental glomerular infarction seenin mock mice were absent in the sG^(b)G mouse kidneys. Infarctions andextramedullary hematopoiesis seen in livers of mock mice were absent inlivers of sG^(b)G mice (FIG. 23G summarizes the data in all groups ofmice). Overall, except for a mild erythroid hyperplasia no organpathology was observed in the sG^(b)G mice.

Example 67 High HbF Expression Improves Survival of Sickle Mice

The life span of BERK sickle mice is significantly reduced, as in humanswith SCA before modern treatment. Kaplan-Meier survival curves showed a100% survival of the sG^(b)G mice at 24 weeks, in contrast to 20%survival in mock mice (n=14, P<0.001).

Example 68 Minimal Parameters Required Correction of SCA

Myeloablative conditioning allows noncompetitive repopulation ofgene-corrected donor HSCs, resulting in high transgene-modified HSCengraftment and transgene expression. It was hypothesized that highlevels γ-globin expression achieved by myeloablative conditioning maynot be necessary for correction, and if so, would reducetransplantation-related morbidity.

Reduced-intensity transplantation was accomplished by transplantinggene-modified BERK LSK cells into sublethally irradiated, but withsignificantly high radiation dose, BERK mice. The proportion oftransduced HSCs and vector copy/cell in the graft was varied bytransducing LSK cells with at a range of MOI (30-100). Since thehalf-life of BERK RBCs was 1.5 to 2 days (FIG. 24G-H), mice weretransfused in the peritransplantation period and analyzed after 12weeks. Three serial experiments were carried out with mice followed for1 year. sG^(b)G mice were analyzed by separating them into 3 groupsbased upon percentage of HbF at 18 weeks: HbF=0% (mock, n=4), HbF lessthan 10% (termed sG^(b)G<10; n=17), and HbF of 10% or more (termedsG^(b)G≧10; n=9); (FIG. 24A). The cutoff at 10% HbF was selected as thisappeared to be a threshold level of HbF that reflected correction ofdisease: sG^(b)G<10 mice showed a higher mortality and inconsistenthematologic correction, compared with sG^(b)G>10 described in thefollowing paragraph. The mouse numbers in the groups changed with timeprimarily due to the increased mortality related to SCA in mice withno/low HbF. The sG^(b)G≧10 group of mice had 16% (±1.2%), 17% (±1.8%),and 21% (±2.3%) HbF, whereas the sG^(b)G<10 group of mice had 5%(±1.4%), 4% (±0.6%), and 4% (±0.5%) HbF at 12, 18, and 24 weeks,respectively, that was stable up to 1 year (FIG. 24B). F-cellrepopulation was significantly higher in sG^(b)G≧10 mice (65%±14%)compared with sG^(b)G<10 mice (30%±9.4%; (FIG. 24C). sG^(b)G≧10 mice had2 to 2.5 vector copies/cell, whereas the sG^(b)G<10 mice had 1.4copies/cell (FIG. 24D).

Example 69 Hematologic Improvement Occurred with Reduced-IntensityTransplantations

Hematologic parameters stabilized at 18 weeks, due to persistenttransfused RBCs in the early posttransplantation period. There was asignificant improvement in hematologic parameters in the sG^(b)G≧10group of mice (FIG. 23G), in contrast to a small and inconsistentimprovement in sG^(b)G<10 mice.

Example 70 Improvement in RBC Function Occurs with Reduced-IntensityTransplantations

Sickling: There was a very significant reduction in ISCs in sG^(b)G≧10mice (P<0.005) and a small, but significant reduction in ISCs insG^(b)G<10 mice compared with mock/BERK controls (P<0.05, FIG. 24E).RBCs from sG^(b)G≧10 mice showed reduced sickling when exposed to gradedhypoxia, compared with RBCs from sG^(b)G<10 or mock/BERK mice (n=20,P<0.01; FIG. 24F). In contrast, there was no significant difference insickling between sG^(b)G<10 and mock/BERK mice. (2) RBC membranedeformability: Surprisingly, despite similar degree of sickling withhypoxia in RBCs from sG^(b)G<10 mice and mock/BERK mice, there wasslight improvement in RBC deformability in the sG^(b)G<10 mice. However,these differences were not statistically significant from the mock/BERKmice due to the high variance (FIG. 24G). In contrast, there was aconsistent significant improvement in RBC deformability in sG^(b)G≧10mice (P<0.001, FIG. 24H). The deformability pattern suggested improvedRBC flow through large vessels and microvessels. (3) RBC survival: RBChalf-life of BERK mice was 1.5 days. RBCs of sG^(b)G mice with 1%, 3%,and 7% HbF had a slightly higher half-life (2 days). Two sG^(b)G micewith 18% HbF showed an RBC half-life of 6 days, a 4-fold increase,similar to that seen in mice carrying 40% HbF in the myeloablativetransplantation model.

Taken together, the sG^(b)G vector resulted in significant andconsistent hematologic and functional correction of SCA, when the HbFproduction exceeded 10% of the total hemoglobin. Notably, theimprovement in phenotype was comparable with that achieved withmyeloablative conditioning.

Example 71 Remarkable Improvement in Organ Pathology when HbFConcentrations Exceed 10%

One unique feature of this BERK→BERK transplantation model was presenceof significant sickle pathology in recipients at the time oftransplantation (determined using BERK controls of comparable age asrecipient mice when they underwent transplantation). Therefore, thepotential for reversal of organ pathology after gene therapy could beassessed. Organ pathology in the surviving mice at approximately 50weeks after transplantation was compared with 3-month-old BERK mice thatdid not undergo transplantation (FIG. 25A; FIG. 25C). The sG^(b)G<10group of mice showed slight improvement in organ pathology: There was aslight reduction in spleen weight (717±162 mg in sG^(b)G<10 vs 870±71 mgin BERK/mock mice; P value, NS). Bone marrow and spleens showed moderateto severe erythroid hyperplasia; livers had infarctions andextramedullary hematopoiesis; and the kidneys showed occasional focalsegmental lesions, focal tubular atrophy, and vascular congestion (FIG.25D). In contrast, a dramatic reversal of organ pathology was seen insG^(b)G≧10 mice: there was a 50% reduction in spleen weight to 363 plusor minus 85 mg, preservation of splenic follicles, and mild erythroidhyperplasia in bone marrow and spleen. Remarkably, no liver infarctionsand no kidney pathology were detected, except in one mouse with a singlefocus of focal tubular atrophy. Overall, sG^(b)G≧10 mice showedcorrection of organ pathology. The lack of organ pathology in sG^(b)Gmice at 15 months of age compared with 3-month-old BERK controlsdemonstrates that gene therapy with the sG^(b)G vector in areduced-intensity transplantation setting prevents any further organdamage, and the existent organ damage at the time of transplantationprobably reverses from regeneration.

Example 72 Survival

There was a significant improvement in overall survival in thesG^(b)G≧10 mice compared with sG^(b)G<10 or mock mice (FIG. 25B;P<0.05). Indeed, at 24 weeks, survival of the sG^(b)G≧10 mice wascomparable with survival in mice with approximately 40% HbF in themyeloablative transplantation model that were followed for 24 weeks.There was some improvement in early survival in sG^(b)G<10 mice comparedwith mock mice (P<0.05). However, by 1 year, there was no difference insurvival of sG^(b)G<10 mice over mock mice.

Example 73 F Cells and HbF/F Cell Critical for Improved RBC Survival andCorrection of SCA

Using biotin surface labeling and intracellular HbF staining, thesurvival of F cells and non-F cells was studied in the same animal,which allowed quantification of the HbF/F cell necessary for improvedsickle RBC survival and deformability. F cells showed a selectiveprolonged survival, as anticipated (FIG. 26A). The average HbF/F cell20in sG^(b)G mice in the BERK3C57B1/6 model was 64% (in these mice, HbFwas 41%±5%, F cells were 64%±6%). In the reduced-intensitytransplantation model, sG^(b)G 10 mice had 32% HbF/F cell (in these miceHbF was 21%±2%, F cells were 65%±14%), and sG^(b)G<10 mice had 13% HbF/Fcell (HbF, 4%±0.1%; F cells, 30%±9.4%). Note that sG^(b)G mice in themyeloablative model and sG^(b)G≧10 mice had similar F-cell repopulation(64%-65%), suggesting that 32% HbF/F cell was sufficient to correct thesickle phenotype. However sG^(b)G<10 mice with 13% HbF/F cell and 30% Fcells had inconsistent and insignificant amelioration of the diseasephenotype.

Therefore, the half-life of F cells in mice was determined, grouped bythe percentage of HbF/F cell. sG^(b)G mice with low (16%; n=2),intermediate (33%; n=4), and very high (89%; n=2) HbF/F cell wasinjected with biotin and followed by periodic blood sampling. It wasdetermined that mice with low HbF/F cell had no improvement in RBChalf-life over BERK controls (FIG. 26B), those with 33% HbF/F cell had a3- to 4-fold improvement in half-life, and mice with very high amountsof HbF/F cell showed RBC survival similar to normal mice. These datademonstrate that if one-third of the hemoglobin within a sickle RBC isHbF, there is significant improvement in RBC survival. Mice with theselevels of HbF/F cell showed approximately 65% F cells, more than 10%HbF.

To confirm the impact of percentage of circulating F cells on overallRBC deformability, mice from both the myeloablative andreduced-intensity experiments (n=34) were grouped into 3 groups: micewith less than 33% circulating F cells, 33% to 65% F cells, and 66% ormore F cells and measured RBC deformability. Only data from the low (3Pa) and high (28 Pa) shear rates are plotted in FIG. 26C. Mice with morethan 66% F cells had a highly significant improvement in RBCdeformability at both high and low shear stress (P<0.01). Mice with 33%to 66% F cells had significantly improved RBC deformability only at highshear stress (P<0.05). Mice with less than 33% F cells showedinconsistent improvement in RBC deformability at low or high shearstress, which was not significantly different from mock controls. Thesedata quantify the critical amount of HbF/F cell, the proportion of Fcells, and overall HbF that are necessary for correction of sickle celldisease.

Example 74 Proportion of Transduced HSCs Required for PhenotypicCorrection

The proportion of HSCs transduced with sG^(b)G in sG^(b)G mice wasanalyzed by the secondary spleen colony-forming unit (CFU-S) assayperformed at 6 months in both models (FIG. 27A-B). Bone marrow aspirateswere performed at 6 months in the BERK→BERK mice that were followed for1 year. The proportion of transduced CFU-S's was determined by HbFexpression. It has been previously shown that all vector-positive CFUsexpress the transgene in an identical vector that encodes β-globin.sG^(b)G mice in the myeloablative conditioning group had 16% to 87%sG^(b)G-transduced CFU-S's (average HSC transduction was ˜50%), andthose in the reduced-intensity group had 5% to 60% transduced HSCs(average HSC transduction was ˜30%). It is to be noted that in thereduced-intensity model, HSC transduction is overestimated, secondary tothe higher mortality of sG^(b)G<10% mice in the first 6 months.

Importantly, 3 mice with 16%, 20%, and 22% transduced CFU-S's had morethan 10% HbF (HbF was 20%, 11%, and 18%, respectively) and showedcomplete phenotypic correction. A vector copy number analysis wasperformed concurrently at 24 weeks on bone marrow cells and showed 1 to3 copies/cell and 1 to 2.5 copies/cell in sG^(b)G mice that underwenttransplantation using the myeloablative conditioning andreduced-intensity conditioning models, respectively. When corrected forHSC transduction, there were 1.5 to 5 vector copies/cell.

Example 75 Transduction of Human CD34⁺ Cells

The percentage of gene-modified HSCs necessary for effective genetherapy is critical in this disease. In vitro studies on SCA marrow canbe done only on a small scale, and would read out correction inprogenitors, not HSCs. HSC correction was shown in humanized models ofSCA with long-term analysis. The extremely limited numbers of RBCsproduced from injecting human thalassemia bone marrow CD34⁺ cells areprohibitive for studies on sickling Therefore, lentivirus transductioninto normal human CD34⁺ cells was optimized for a preclinical scale-up,using a GFP lentivirus vector and the severe combined immunodeficient(SCID)-repopulating assay. Granulocyte colony-stimulatingfactor-mobilized peripheral blood CD34⁺ cells transduced with a GFPlentivirus vector were transplanted into nonobese diabetic(NOD)/LtSz-scid IL2Rynull (NSG) mice. Here, mock mice were those thatreceived a transplant of untransduced CD34+ cells immediately afterselection, as controls for the effect of transduction on engraftment andclonogenicity. At 6 weeks, CFUs were plated from bone marrow derivedfrom NSG mice, and 36 individual CFUs/mouse were analyzed for thepercentage of gene-marked colonies. The 18-hour transduction did notaffect engraftment or clonogenicity (data not shown). A 77% genetransfer on average was observed in the SCID-repopulating cell assay,similar to previous data in human thalassemia CD34⁺ cells.

The data from this study indicates that lentiviral delivery humanγ-globin under β-globin regulatory control elements in HSCs results insufficient postnatal HbF expression to correct SCA in mice. The amountof HbF and transduced HSCs was then de-scaled, using reduced-intensityconditioning and varying MOI, to assess critical parameters needed forcorrection. A systematic quantification of functional and hematologicRBC indices, organ pathology, and life span were critical to determinethe minimal amount of HbF, F cells, HbF/F cell, and gene-modified HSCsrequired for reversing the sickle phenotype.

Results indicate the following: (1) Amelioration of disease occurredwhen HbF exceeded 10%, F cells constituted two-thirds of the circulatingRBCs, and HbF/F cell was one-third of the total hemoglobin in RBCs; andwhen approximately 20% sG^(b)G modified HSCs repopulated the marrow. (2)Genetic correction was sustained in primary or secondary transplantrecipients followed long-term. (3) There is a method of determiningminimum HSC chimerism for correction of a hematopoietic disease in an invivo model, which would contribute to design of cell dose andconditioning regimens to achieve equivalent genetically corrected HSCsin human clinical trials.

One novel aspect of this study is that it addresses, for the first time,the gene dosage and the gene-modified hematopoietic stem cell dosagerequired for correction of a genetic defect. Expressing a tremendousamount of fetal/antisickling hemoglobin will undoubtedly correctdisease, as has been shown by others, but is not practically possible ina clinical setting. As an example, an initial gene therapy for adenosinedeaminase (ADA) deficiency was performed using no conditioning, and wasnot therapeutic, even though few gene-marked stem cells engrafted, and aselective advantage to gene-corrected lymphocytes was evident uponwithdrawal of ADA. In a subsequent trial, 4 mg/kg busulfan was usedbefore transplantation, as conditioning, resulting in adequategene-corrected stem cell dose and gene-modified T cells. Although thesepioneering studies provided us with invaluable information, theyunderscore the critical importance of determining thresholds for geneticcorrection before embarking on clinical studies.

Although disease has been corrected at 1 to 3 copies/cell, the presentstudy indicates that the percentage of transduced stem cells in thissetting of lethal irradiation/transplantation is very high (average HSCstransduced are 50%, as analyzed by a stringent secondary CFU-S assay).This level of HSC transduction would likely not be achieved in theclinical setting unless myeloablation is performed.

Therefore a novel model (BERK to BERK transplantation) was developed toaddress the minimal gene transfer needed, and answer questions ofcorrection of SCA in a mouse with significant sickle pathology at 12weeks of life (FIG. 25). Notably, a sickle to normal myeloablativetransplantation, used by other groups showing correction of SCD, is adisease prevention model, where there was no underlying pathology attime of transplantation. The present studies show that repair ofpreexisting pathology can occur, if genetic correction results in morethan 10% HbF.

BERK mice have some degree of thalassemia. Therefore one concern inusing this model for genetic therapy studies for sickle cell anemia isthat correction of thalassemia would obscure improvements made by theantisickling effects of HbF. Surprisingly no significant change in MCHin sG^(b)G<10 or sG^(b)G≧10 mice, including mice with HbF/F cell as highas 89% were seen (as disclosed herein). These results were surprising,but showed that the correction of sickling in RBCs was not secondary tocorrection of thalassemia, as seen in murine thalassemia model, whereincreasing MCH was seen with increases in HbF of 4% or higher.Conceivably, HbF is produced at the expense of HbS.

Although BERK mice exclusively carry human hemoglobin, the totalhemoglobin in the mouse RBCs is one-third of a human RBC. Therefore, HbFand HbF/F cell were expressed as a percentage, rather than in absoluteamounts, to best compare murine data to human. An increase of HbF from3.6% to 13.6% has been shown to reduce acute sickle events in patientson decitabine. Similar improvement in sickle events occurs with 25% ormore HbF/F cell in patients responsive to hydroxyurea. Data presentedhere, indicating improvement with 33% HbF/F cell, is concordant withthese reports, but more closely resemble RBCs in infants with SCA, whereless than one-third HbF/F cell at 10 to 12 months is considered athreshold for intracellular sickle polymerization. The most remarkableeffect of γ-globin production with the sG^(b)G vector was a dramaticabsence of chronic organ damage and an improved survival of the sicklemice when HbF exceeded 10%. Patients with high HbF have an improvedsurvival, confirmed by the multicenter study on hydroxyurea. HbFexpressed from the sG^(b)G vector was comparable with, or even betterthan, effective hydroxyurea treatment. The potential of a one-timecorrection, where responsiveness to hydroxyurea and compliance to dailylife-long administration would not be limiting factors, would be atremendous advantage of gene therapy. Indeed, we did not anticipate wewould get the same conclusion with gene therapy, as derived fromcollective knowledge on (1) transgenic mice, in which every RBC has thesame amount of HbF although we were imposing HbF on SS RBCs; (2)chimeric transplantations, in which normal amounts of HbA-producing RBCs(AA RBC) are present mixed with SS RBCs17,37,38; or (3) SCD patients onhydroxyurea, in whom macrocytosis induced by hydroxyurea would diluteHbS and reduce the threshold for sickling. A much higher threshold ofgenetically corrected sickle HSCs necessary for F-cell repopulation andcorrection of SCA phenotype was expected, as HbF was exogenously imposedinto a sickle cell with normal amounts of HbS. Notably, despite thesedistinct differences in transgenics/chimeras, conclusions were similarwith exogenous γ-globin expression: Indeed expressing exogenous HbF inRBCs at concentrations from 33% to as high as 89% resulted in nosignificant increase in MCV or MCH, yet corrected sickling. This datasuggests that genetic delivery of HbF decreases endogenous HbS.

The percentage of transduced HSCs in the setting of lethalirradiation/transplantation is very high (50% on average, as analyzed bya stringent secondary CFU-S assay at 24 weeks), a number that would bedifficult to achieve in a clinical setting. The BERK→BERKtransplantation model, however, shows that 20% autologous HSC correctionmay suffice for a significant amelioration of sickling, organ damage,and survival. However, whether this percentage of gene-modified HSCsnecessary for effective gene therapy is achievable is critical todetermine, since there is no survival advantage to the gene-modifiedHSCs in this disease. High human HSC transduction has been a limitationof gene therapy with the traditional gamma retrovirus vectors.Lentivirus vectors can overcome this barrier: a 20% long-termtransduction has been shown in adrenoleukodystrophy with a lentivirusvector. Lentivirus transduction into human CD34+ cells was optimized,using the SCID-repopulating cell assay and achieved approximately 75%gene transfer in SCID-repopulating cell, on average, similar to previousdata in human thalassemia CD34+ cells, where 70% transduction was seen 3to 4 months after transplantation into immune-deficient mice. Notably,this level of gene transfer in the SCID mice is encouraging, and indeedhigher than the gene transfer observed in NOD-SCID mice with theadrenoleukodystrophy lentivirus vector in preclinical studies.

Gene therapy using this approach could also overcome the toxicity andimmunologic consequences of the traditional allogeneic bone marrowtransplantation/reduced-intensity transplantation. Mismatched mixedchimerism of normal and sickle marrow in murine transplantations showsthat a near complete chimerism is typically necessary for correction oforgan damage. It is encouraging that, in a clinical series,reduced-intensity conditioning (RIC) transplantation with 8 mg/kgbusulfan along with fludarabine, antithymocyte globulin, and totallymphoid irradiation in SCA patients has shown an average allogeneicengraftment of 78% at 2 to 8.5 years after transplantation, withcorrection of SCA phenotype. This high level of donor chimerism even inan allogeneic RIC setting, where immune rejection can occur, suggeststhat high gene transfer efficiency into autologous CD34+ cells followedby RIC may be a potentially safer alternative to myeloablativeconditioning. 77% gene transfer efficiency in human stem/progenitors wasdemonstrated using a NOD-SCID repopulating cell assay, as well acorrection of phenotype in mice with 1.3 to 1.5 copies per cell andapproximately 20% gene-marked CFU-Ss (FIG. 27).

Significantly, correction occurred at 1 to 3 vector copies per cell, aclinically achievable goal. Flanking the sG^(b)G virus with a chromatininsulator is expected to increase HbF/vector copy by 2- to 4-fold. Inexperimental models, the insulator appears to reduce clonal dominance,although whether the insulator element lowers the risk of insertionaloncogenesis is unknown. The risk of insertional oncogenesis observedwith randomly integrating vectors has been shown to be lower with alentivirus vector than a gammaretrovirus vector. It would be furtherlowered when the enhancer element is active only in a restrictederythroid lineage

Example 76 Gene Therapy for Sickle Cell Disease

Since HbF is the hemoglobin with the highest anti-sickling effect, alentivirus vector, sG^(b)G, that carries a normal human γ-globin genewas used to produce HbF in Berkeley sickle mice. As disclosed herein, alentivirus vector incorporating γ-globin exons and β-globin non-codingregions and regulatory elements, sG^(b)G, was designed. This vectorshowed complete correction of the sickle phenotype in Berkeley sicklemice following transfer of the sG^(b)G vector into HSCs andmyeloablative transplants (Example 65, FIGS. 23A-D, FIG. 28, and Table1). FIGS. 23A and B show the reduction in irreversibly sickled cells inblood of the mice, FIGS. 23C and D show experimentally induced sicklingof RBC from the mice and the proportion of sickled cells, and FIG. 28shows the improvement in survival of mice following successful genetherapy.

TABLE 1 Hematological correction is obtained in the sG^(b)G group ofBerkeley sickle mice; correction is sustained long-term in primary andsecondary transplants. Mice N WBC RBC Hb MCV MCH RDW Plt Retic (% BERK 556.8 ± 5.4 5.3 ± 0.4  5.8 ± 0.5 48.2 ± 1 10.7 ± 0.5 35.3 ± 1.6 733 ± 8060.8 ± 5.0 sGbG Prim. 5 10.6 ± 3.1 9.4 ± 0.8 10.0 ± 0.8 40.7 ± 1 10.4 ±0.6 27.6 ± 1.1 733 ± 82 15.8 ± 3.2 Mock Prim. 10 29.7 ± 1.4 5.8 ± 0.4 7.6 ± 0.7 48.5 ± 1 10.7 ± 0.2 32.0 ± 0.9 921 ± 50 40.0 ± 3.0 P value*0.001 0.007 0.03 0.001  0.9 0.009 0.06 0.006 sG^(b)G Sec. 6  6.8 ± 1.48.9 ± 0.4 10.1 ± 0.5 40.5 ± 2 11.4 ± 0.5 28.3 ± 1.4 658 ± 33 13.8 ± 2.9Mock Sec. 1 31.7 5.2  6.4  47.6 12.2 32.1 923 49 Analysis of bloodparameters of mice is shown 18 weeks following primary (Prim.) andsecondary (Sec.) transplants of sG^(b)G or Mock transduced lineage (−),Sca (+) and Kit (+) cells.

The critical parameters necessary to correct SCA pathophysiology using areduced intensity transplant were then determined. Complete correctionof the hematological and functional RBC parameters, inflammation, andorgan pathology was observed in SCD mice followingmyeloablative-conditioning and transplant. Correction was sustainedlong-term in primary and secondary transplant recipients. The criticalparameters necessary to correct SCA pathophysiology using a reducedintensity transplant were also determined. There was 100% 6-monthsurvival of genetically-corrected Berkeley sickle mice, compared to 20%survival of mock-transplanted Berkeley sickle mice.

Using reduced-intensity conditioning and simulating conditions ofautologous transplant, different proportions of gene-modified Berkeleysickle HSC were transplanted into sub-lethally irradiated Berkeleysickle mice. The minimal proportions of genetically-corrected HSC, HbF,HbF-containing RBC (F-cells) and HbF/F-cell required for correction ofsickle cell anemia were then defined. With 15-20% gene modified HSCrepopulating the Berkeley sickle mouse bone marrow, approximately 2vector copies per cell, >10% HbF, and >66% F cells, there was completecorrection of the sickle phenotype, including organ pathology andsurvival. With 15-20% gene-modified HSCs repopulating the Berkeleysickle mouse bone marrow, approximately 2 vector copies per cell, >10%HbF, and >66% F cells, there was complete correction of the sicklephenotype, including organ pathology and survival.

Example 77 Gene Therapy for Beta Thalassemia

Expression of HbF via lentivirus vectors carrying the human γ-globingene has been previously shown (Persons et al. Blood 10:2175-83 (2003);Pestina et al. Mol. Ther. 17:245-52 (2009)). In order to confirm theability of the sG^(b)G vector to correct β-thalassemia, thalassemia mice(Hbbth3/+) were treated using the same approach as used in sickletransgenic mice. Thalassemia mice were transplanted with thalassemiastem/progenitor cells (Lin− Sca+ Kit+ [LSK] cells) transduced twiceabout 8 hours apart with sG^(b)G (MOI of 2×20). Control (Mock) animalswere concurrently treated with media only. Approximately 10,000transduced LSK+ cells were injected/co-transplanted with 200,000irradiated Lin-Sca-Kit− cells into lethally irradiated thalassemiarecipient mice (split dose of 700+375 rads). The primary animals weremonitored over a period of 7-8 months, and secondary transplants wereperformed thereafter for a total follow up of 18 months.

The vector resulted in increased HbF to 22±3% (mean±SEM); whichcorrected the thalassemia phenotype (FIG. 29). There was a rise inhemoglobin from a mean of 8.8±0.2 g/dL in mock transplanted mice to12.5±0.5 g/dL in sG^(b)G transplanted mice (FIG. 29A); hematocrit rosefrom 31.8±0.3 to 42.1±1.07 (FIG. 29B). This was accompanied by a fall inreticulocytosis from 20.8±0.3% to 8.7±1.4% (FIG. 29D). The microcytosisseen in thalassemia was also corrected, with an increase in MCV from38.1±0.3 to 45.3±1.7 fl. This correction was stable over time and wassustained in primary and secondary mice.

Example 78 Improved HbF Expressing Vector with Superior Anti-SicklingProperties

As described above, production of >10% HbF was shown to correct the SCDphenotype in the mouse model. While the sG^(b)G vector efficientlycorrected the phenotype in the Berkeley sickle mouse model (Example 65,FIGS. 23A-D, FIG. 28; Perumbeti et al., Blood 114:1174-85 (2009)), itwas far less efficacious in the knock-in sickle mouse model (UAB mice),unless very high vector copies were present per cell.

The Berkeley sickle mice are transgenic for the human α- andβ^(S)-globin transgenes, and knock-out of mouse globins and the humantransgenes leads to imbalanced globin chain production. Berkeley sicklemice have a relative excess of human α globin chains, as compared toβ^(S) globin chains, allowing the γ-globin produced by the sG^(b)Gvector to form HbF (α₂γ₂) readily, in the presence of β^(S) globin thatalso binds a-globin to form HbS (α₂β^(S) ₂). The UAB mice, on the otherhand, are knock-ins for human α in place of the mouse α globin and humanβ^(S) in place of the mouse β^(major) globin gene, producing humanglobins in place of mouse globins. These mice therefore have completelybalanced human α and β^(S) chains and resemble patients with homozygoussickle cell anemia (Hb SS disease). Patients with homozygous SCD (andthe UAB knock-in sickle mouse model) have balanced (equal amounts of) αand β^(S) molecules, and the genetically introduced γ-globin has tocompete with β^(S) for a-globin. Hence, a far excess of γ-globin isrequired to outcompete β^(S) globin to form HbF.

Accordingly, in Berkeley sickle mice, γ-globin produced by this vectoreffectively binds the excess a globin to form HbF, without muchcompetition from β^(S) globin, which binds with a globin to form HbS.Hence, in Berkeley sickle mice, at clinically achievable vector copies,correction of disease is observed. However, in UAB mice, the a globinchains become rate limiting due to equal amounts of competing β^(S)globin chains. Therefore, a high level/excess of vector-derived γ globinis required to be able to out compete β^(S) globin to form HbF.

To address this, several changes were made to the gene transfer protocoland strategy. The γ-globin gene was modified with a GA point mutation atbp 50 in exon 1. This modification changes the amino acid glycine (GGC)to aspartic acid (GAC) in order to improve its affinity for a-globinwithout altering its functional properties, so that HbF is formed athigher efficiency than HbS in RBCs. The ability of the original γ globinvector (sG^(b)G) was then compared to that with a point mutation in theγ globin coding sequence (sG^(b)G^(M)) in sickle mice. The annotatedvector map for the sG^(b)G^(M) is depicted in FIG. 30, with the variousregions of the sequence identified in FIGS. 30 and 31. The sG^(b)G^(M)sequence is shown in FIG. 31.

Comparative studies between the sG^(b)G and sG^(b)G^(M) vectors weredone in Berkeley sickle mice and in UAB sickle mice, where lineagedepleted, Sca+ Kit+ (LSK) cells, that are highly enriched inhematopoietic stem cells, were sorted, transduced with medium alone(mock), the sG^(b)G vector, or the sG^(b)G^(M) vector, and transplantedinto sub-lethally irradiated Berkeley or UAB sickle recipient mice, aspreviously described for the sG^(b)G efficacy studies (Perumbeti et al.,Blood 114:1174-85 (2009)). Mice were bled at 6, 12, and 24 weekspost-transplant to assess hematological parameters and HbF expression,and six to eight mice per arm were then followed for 6-12 months. The12-week data in Berkeley sickle mice are shown in FIG. 32, whichdemonstrates the superior HbF production per vector copy and improvedreticulocytosis from the sG^(b)G^(M) vector.

Comparative results between Berkeley and knock-in UAB sickle mice areshown in FIG. 33. The data shown are results from a 6-month analysis.The amount of HbF produced per vector copy from the sG^(b)G andsG^(b)G^(M) vectors is shown in Berkeley sickle mice (FIG. 33A) and UABknock-in sickle mice (FIG. 33B). The sG^(b)G^(M) mice showed nearly1.5-2 times superior ability to form HbF as compared with the sG^(b)Gvector in both types of sickle mice. It is notable that the amount ofHbF produced per copy of the sG^(b)G^(M) vector is nearly twice in theBerkeley mice when compared to UAB mice with the sG^(b)G^(M) vector,showing the ease with which HbF tetramers form if excess a-globin chainsare present and the difficulty forming these tetramers if a-globinchains are rate limiting.

The HbF generated from the sG^(b)G^(M) vector was functional, showingcorrection of sickling, a superior reduction in reticulocytosis, and arise in hemoglobin in both types of sickle mice. Correction of anemiawas observed in UAB mice transplanted with the sG^(b)G^(M) vector butnot the sG^(b)G vector. Some of these mice have now been followed fornearly one year, and the expression is stable. Much better correction ofRBC half-life and RBC membrane deformability was also observed with thesG^(b)G^(M) vector as compared to the sG^(b)G vector when HbF levels arethe same. FIG. 32 shows that despite HbF levels of 30-35% with thesG^(b)G vector (filled diamonds, FIG. 32B), mice had an averagereticulocyte count of approximately 15%, while the reticulocyte countwas 5% in sG^(b)G^(M) mice with similar HbF levels (open diamonds). Thisdemonstrates that the mutation also improved the lifespan of the RBCs byreducing sickling, despite the similar levels of HbF. Thus, theengineered mutated gamma globin vector also produces a superioranti-sickling HbF, improving RBC quality and lifespan. Studies relatingto the effect on organ damage, RBC membrane deformability, RBChalf-life, and the oxygen affinity of the mutant HbF compared to normalHbF to determine the mechanism behind this unexpected favorable propertyof HbF are ongoing.

These results demonstrate that the point-mutated γ-globin gene in thesG^(b)G^(M) vector prevents sickling and therefore prolongs sickle RBChalf-life, leading to lower reticulocyte counts (FIG. 33). This higherproduction of HbF and reduction in reticulocytosis results in aproportional rise in hemoglobin and hematological correction of thesickle phenotype. The sG^(b)G^(M) vector has no change in the vectorbackbone or any of the transcriptional regulatory elements.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the specific number of genesor targeted by a therapeutic product, the type of gene, the type ofgenetic disease or deficiency, and the gene(s) specified. Variousembodiments of the invention can specifically include or exclude any ofthese variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

What is claimed is:
 1. A mutated human gamma-globin gene, wherein themutated human gamma-globin gene encodes a protein comprising SEQ IDNO:1.
 2. The mutated human gamma-globin gene, wherein the mutated humangamma-globin gene has a sequence identity of 70% or greater to SEQ IDNO:
 2. 3. A method of using the mutated human gamma-globin gene of claim1 to genetically correct sickle cell anemia or β-thalassemia or reducesymptoms thereof, the method comprising: identifying a subject in needof treatment for sickle cell anemia or β-thalassemia; transfectingautologous hematopoietic stem cells (HSCs) with a modified lentiviruscomprising the mutated human gamma-globin gene of claim 1; andtransplanting the transfected HSCs into the subject.
 4. The method ofclaim 3, wherein the subject is a human subject.
 5. The method of claim3, further comprising treating the subject with reduced intensityconditioning prior to transplantation.
 6. The method of claim 3, whereinthe modified lentivirus further comprises a heterologous polyA signalsequence downstream from a viral 3′ LTR sequence in a standard SINlentiviral vector backbone; and one or more USE sequences derived froman SV40 late polyA signal in a U3 deletion region of a standard SINlentiviral vector backbone.
 7. The method of claim 6, wherein themodified lentivirus further comprises one or more flanking CHS4-derivedreduced-length functional insulator sequences.
 8. The method of claim 7,wherein the modified lentivirus further comprises a beta-globin locuscontrol region.
 9. The method of claim 7, wherein the modifiedlentivirus further comprises an erythroid lineage specific enhancerelement.
 10. The method of claim 3, wherein: post-transplantation fetalhemoglobin exceeds at least 20%; F cells constitute at least 2/3 of thecirculating red blood cells; fetal hemoglobin per F cells account for atleast 1/3 of total hemoglobin in sickle red blood cells; and at least20% gene-modified HSCs re-populate bone marrow of the subject.
 11. Alentiviral expression vector capable of genetically correcting sicklecell anemia or β-thalassemia or reducing symptoms thereof, comprisingthe mutated human gamma-globin gene of claim
 1. 12. The lentiviralexpression vector of claim 11, further comprising: a heterologous polyAsignal sequence downstream from a viral 3′ LTR sequence in a standardSIN lentiviral vector backbone; and one or more USE sequences derivedfrom an SV40 late polyA signal in a U3 deletion region of a standard SINlentiviral vector backbone.
 13. The lentiviral expression vector ofclaim 12, further comprising one or more flanking CHS4-derivedreduced-length functional insulator sequences.
 14. The lentiviralexpression vector of claim 13, further comprising one or more elementsof a beta-globin locus control region cloned in reverse orientation to aviral transcriptional unit.
 15. The lentiviral expression vector ofclaim 13, further comprising an erythroid lineage specific enhancerelement.